Engineered cells secreting therapeutic enzymes

ABSTRACT

Provided herein are mammalian cells comprising a first exogenous nucleic acid encoding a DNAse protein and a second exogenous nucleic acid encoding another DNAse protein, such as DNASE1 protein and DNASE1L3 protein, that have improved properties, including the ability to degrades extracellular chromatin and remove Neutrophil Extracellular Traps (NETs). Use of these cells, including the use in the treatment of a subject in need thereof, is also contemplated.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.provisional patent application, U.S. Ser. No. 63/010,489, filed Apr. 15,2020, and U.S. provisional patent application, U.S. Ser. No. 63/047,516,filed Jul. 2, 2020, the entire content of both of which are incorporatedherein by reference.

BACKGROUND

Neutrophil Extracellular Traps (NETs) are extracellular webs ofchromatin, i.e., DNA and associated nuclear proteins, released byneutrophils in a controlled process called NETosis. NETs were discoveredonly 16 years ago but have since been recognized as a fundamental andbiologically ancient component of innate immunity and host defense. SeeNeumann A. et al., Extracellular traps: an ancient weapon of multiplekingdoms, Biology 2020, doi 10.3390/biology9020034; Burgener S. et al.,Neutrophil Extracellular Traps in host defense, Cold Spring HarbPerspect Biol. 2019, doi 10.1101/cshperspect.a037028.

NETs have also been implicated as key mediators in a wide array of acuteand chronic diseases, e.g., acute respiratory distress syndrome (ARDS),acute kidney injury (AKI), sepsis, myocardial infarction, systemic lupuserythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SS),asthma, and various cancers. See, e.g., Keyel P. A., DNAses in healthand disease, Dev. Biol. 2017, doi 10.1016/j.ydbio.2017.06.028; Fuchs T.A. et al., Neutrophil Extracellular Traps, Inflammation 2018, doi10/1142/9789813198445_0006.

There has been much interest in therapies to remove NETs to treat orprevent human disease. One such therapy is the therapeuticadministration of exogenously produced DNAse enzymes, e.g., DNAse 1(DNASE1) or DNAse 1-like-3 (DNASE1L3), to an individual in need thereof.See, e.g., U.S.S.N. 2014/0199329 and U.S.S.N. 2020/0024585

There are many challenges and shortcomings, however, to the manufactureand administration of exogenously produced DNAse enzymes. For example,such enzymes are difficult to manufacture, not least because they can betoxic to cells customarily used for recombinant expression. Most DNAseenzymes are rapidly eliminated from the systemic circulation; and theycan be immunogenic.

Therefore, a new approach for the therapeutic removal of NETs.

SUMMARY OF THE INVENTION

The present invention arises from the inventors' discovery that amammalian cell, e.g., a human Natural Killer (NK) cell or MesenchymalStem (or Stromal) Cell (MSC), can be modified in vitro to secrete one ormore DNAse enzymes that remove NETs, and that such a cell can beadministered to an individual in need thereof for therapeutic orprophylactic purposes. The DNAse enzymes can be, e.g., DNASE1L3, DNASE1,another DNAse, or a combination thereof. There are several potentialbenefits to this approach. For example, with reference to MSCs: (1) MSCscells naturally home to sites of inflammation within the body, such asthe lungs in patients with Acute Respiratory Distress Syndrome (ARDS).Thus, in contrast to systemic administration of an enzyme, whichprovides uniform enzyme concentrations in the bloodstream, MSCs willdeliver the enzyme in close proximity to the inflamed tissue where NETsare concentrated. (2) MSCs can be produced from a separate donor forsafe “off-the-shelf” administration to one or more recipients differentfrom the donor, without risk of an adverse host immune response. Thismakes it possible to manufacture the cells economically, in bulk, and inadvance, so that they can be immediately available when needed. (3) TheMSCs can be modified to express and secrete one or more native DNAseenzymes by introduction of recombinant RNA or DNA, and the resultantDNAse enzyme(s) will have all the normal post-translationalmodifications that render them identical to the native enzyme. Thisstands in contrast to the usual industrial production of DNAse enzymesin E. coli, yeast, insect, or mammalian cell lines, which is not onlycostly and labor-intensive, but can also yield products that differ fromthe native enzyme, for example, due to differences in post-translationalmodification. These potential benefits are meant to be non-limiting andthe disclosed invention would still be beneficial without one or more ofthese potential benefits.

The invention also embraces other cells, including, but not limited to,homospecific cells, e.g., blood cells (e.g., T cells, NK cells,monocytes, macrophages or CD34+ cells), or other stem cells, modified invitro to produce one or more DNAse enzymes to remove NETs.

Where the invention describes cells modified to secrete one or moreDNAse enzymes, it is also envisioned that any of those cells may befurther modified to express one or more additional proteins. Forexample, the cells described herein can be further modified to expressan anti-BCMA protein. B cell maturation antigen (BCMA) is a tumornecrosis family receptor (TNFR) member expressed in cells of the B celllineage. BCMA expression is the highest on terminally differentiated Bcells, e.g., plasma cells. BCMA is involved in mediating the survival ofplasma cells for maintaining long-term humoral immunity. Therefore,BCMA-positive cells also play an important role in certain cancers,e.g., myeloma and Hodgkin lymphoma, and diseases mediated byauto-antibodies, e.g., myasthenia gravis, systemic lupus erythematosus,rheumatoid arthritis, blistering skin diseases (e.g., pemphigus,psoriasis), inflammatory bowel disease, celiac sprue, pernicious anemia,idiopathic thrombocytopenia purpura, scleroderma, Graves' disease,Sjögren syndrome, Goodpasture syndrome, and type 1 diabetes. In someconditions where both auto-antibodies and NETs play a role, e.g.,systemic lupus erythematosus, the ability to reduce or remove both NETsand BCMA+ cells would be of particular value. Indeed, the inventorsenvision that simultaneous reduction of NETs and BCMA+ cells wouldprovide synergistic benefits in the aforementioned conditions,especially systemic lupus erythematosus. For purposes of thisdisclosure, an “anti-BCMA protein” means a protein that specificallybinds to BCMA. Preferably, the anti-BCMA protein causes reduction ofBCMA+ cells. The anti-BCMA protein can be, e.g., an anti-BCMA monoclonalantibody; or a bispecific antibody, e.g., a bispecific T-cell engager,e.g., directed against each of BCMA and CD3. See, e.g., U.S. Pat. Pub.No. 2019/0263920A1.

Furthermore, the invention includes not only the inventive cells, butalso specific constructs or vectors used in production of the inventivecells, methods to produce the inventive cells, methods of treatment thatcomprise administration of the inventive cells to an individual in needthereof, and uses of the inventive cells for the treatment or preventionof disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a chromatin degradation assay with DNAelectrophoresis following treatment of chromatin with supernatants ofcells transfected with DNASE1 and DNASE1L3, or cells that wereuntransfected. The cells used in this assay were MSCs, CD4+ T cells,CD8+ T cells, and NK cells.

FIG. 2 shows the results of a chromatin degradation assay with DNAelectrophoresis following treatment of chromatin with MSC supernatantsamples. Lane A represents unmodified MSCs. Lane B represents MSCsmodified to express DNASE1. Lane C represents MSCs modified to expressDNASE1L3 (by means of a pseudouridine-substituted mRNA). Lane Drepresents MSCs modified to express both DNASE1 and DNASE1L3.

FIG. 3 shows a series of photographs wherein macroscopic amounts of NETsare degraded by GR-17 supernatants. The photographs show the addition ofNETs, the addition of supernatant, 4 minutes post addition, 8 minutespost addition, and 10 minutes post addition.

FIGS. 4A-4B shows the levels of DNase-encoding mRNA over time in MSCs.GR-17 samples were prepared, frozen, and thawed. MSCs were incubated forup to 6 days (D1 to D-6) in complete media. mRNA was assayed byquantitative RT-PCR using primers specific for DNase1 and DNase1L3.Results are expressed as mRNA copies per total cell RNA. FIG. 4Arepresents DNase1 mRNA. FIG. 4B represents DNase1L3 mRNA.

FIGS. 5A-5B show the level of DNASE1 and DNASE1L3 expression usingWestern blot analysis. GR-17 was prepared, frozen, and thawed. MSCs wereincubated for up to 6 days (D1 to D-6) in complete media. DNase1 andDNase1L3 protein were assayed by Western Blot. FIG. 5A represents DNase1protein expression. FIG. 5B represents DNase1L3 protein expression atDay 1. No expression was evident at Day 2-6.

FIGS. 6A-6D show the results of a chromatin degradation assay using DNAelectrophoresis following treatment of naked DNA, chromatin, and NETs.GR-17 was prepared, frozen, and thawed. MSCs were incubated for up to 6days (D1 to D-6) in complete media. Supernatant was collected at theindicated timepoints and assayed for its capacity to degrade cell-freenaked DNA (FIG. 6A), chromatin (FIG. 6B), and NETs (FIG. 6C). Negativecontrols include water-transfected MSC supernatants at Days 1 and 6 andno-supernatant control. Positive controls are exogenous recombinanthuman DNase1 and purified human DNase1L3. FIG. 6D shows a fluorescentmicrograph of NETs induced from human neutrophils by incubating with 100μg/mL of phorbol myristate acetate (PMA).

FIGS. 7A-7C chart the time- and dose-dependence of naked DNA and NETdegradation by MSCs. Increasing concentrations of GR-17 or control MSCswere cultured overnight in the presence of naked DNA (FIG. 7A) or NETs(FIG. 7B). FIG. 7C shows a representative fluorescence micrograph ofNETs cultured overnight in the presence of 100,000 MSC or 100,000 GR-17(highest concentration). Background image settings are identical betweencontrol and GR-17 conditions and the images are at the samemagnification.

FIG. 8 shows the results of a chromatin degradation assay using DNAelectrophoresis conducted to assess degradation of NETs over time byMSCs. GR-17 or control MSCs were cultured in the presence of exogenousNETs for up to 48 h and assayed for NET degradation at the indicatedtimepoints.

FIG. 9 shows the results of a chromatin degradation assay using DNAelectrophoresis with various MSCs made according to a specificembodiment of the invention. MSCs were transfected with DNase1 mRNA orDNase1L3 mRNA and cultured. Between timepoints (e.g., day 0 to day 1(D1)) supernatants were collected and used in chromatin digestionassays. Control MSCs were transfected with an irrelevant protein.

FIG. 10 shows the results of a chromatin degradation assay using DNAelectrophoresis with MSCs made according to a specific embodiment of theinvention, and in the presence of no serum, 50% serum, or 100% serum.GR-17 was frozen, thawed and incubated in the presence of NETs overnightin media supplemented with 0%, 50% or 100% fresh off-the-clot serumcollected from healthy volunteers. Serum incubation was done inreplicates.

FIG. 11 shows the results of a chromatin degradation assay using DNAelectrophoresis with MSCs made according to a specific embodiment of theinvention. MSCs were transfected with wild-type (U) or pseudouridine (ψ)DNase1 or DNase1L3 mRNA and cultured as described. Between timepoints,e.g., day 0 to day 1 (D1), supernatants were collected and used inchromatin digestion assays.

DEFINITIONS

As used herein and in the claims, the singular forms “a,” “an,” and“the” include the singular and the plural reference unless the contextclearly indicates otherwise. Thus, for example, a reference to “anagent” includes a single agent and a plurality of such agents.

The term “antibody”, as used herein, broadly refers to anyimmunoglobulin (Ig) molecule comprised of four polypeptide chains, twoheavy (H) chains and two light (L) chains, or any functional fragment,mutant, variant, or derivation thereof, which retains the essentialepitope binding features of an Ig molecule. Such mutant, variant, orderivative antibody formats are known in the art.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgA1 andIgA2) or subclass.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen. Ithas been shown that the antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Such antibodyembodiments may also be bispecific, dual specific, or multi-specificformats; specifically binding to two or more different antigens.Multispecific, dual specific, and bispecific antibody constructs arewell known in the art and described and characterized in Kontermann(ed.), Bispecific Antibodies, Springer, NY (2011), and Spiess et al.,Mol. Immunol. 67(2):96-106 (2015).

Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publicationWO 90/05144 A1 herein incorporated by reference), which comprises asingle variable domain; and (vi) an isolated complementarity determiningregion (CDR). Furthermore, although the two domains of the Fv fragment,VL and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. Other forms of single chain antibodies, such as diabodiesare also encompassed. Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Suchantibody binding portions are known in the art (Kontermann and Dubeleds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp.(ISBN 3-540-41354-5).

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations, kappa and lambda light chainsrefer to the two major antibody light chain isotypes.

The term “synthetic antibody” as used herein, refers an antibody whichis generated using recombinant DNA technology, such as, for example, anantibody expressed by a viral vector. The term should also be construedto mean an antibody which has been generated by the synthesis of a DNAmolecule encoding the antibody and which DNA molecule expresses anantibody protein, or an amino acid sequence specifying the antibody,wherein the DNA or amino acid sequence has been obtained using syntheticDNA or amino acid sequence technology which is available and well knownin the art.

An “effective amount” refers to the amount of a therapy which issufficient to reduce or ameliorate the severity and/or duration of adisorder or one or more symptoms thereof, prevent the advancement of adisorder, cause regression of a disorder, prevent the recurrence,development, onset or progression of one or more symptoms associatedwith a disorder, detect a disorder, or enhance or improve theprophylactic or therapeutic effect(s) of another therapy (e.g.,prophylactic or therapeutic agent).

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include, but are not limited to,those known in the art, such as cosmids, plasmids (e.g., naked orcontained in liposomes) and viruses (e.g., lentiviruses, retroviruses,adenoviruses, and adeno-associated viruses) that incorporate therecombinant polynucleotide.

Unless otherwise specified, where used in reference to a nucleic acid ornucleic acid construct, “exogenous” refers to a nucleic acid or nucleicacid construct that originates from outside a cell and is introducedinto the cell by one more artificial manipulations.

An exogenous nucleic acid can include, without limitation, nucleic acidanalogs, unnatural and/or modified nucleotides, and other modificationsknown in the art, including, without limitation, 5′ caps or othercovalently linked chemical moieties known in the art.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

Unless otherwise specified, a “nucleotide sequence or nucleic acidencoding an amino acid sequence” includes all nucleotide sequences thatare degenerate versions of each other and that encode the same aminoacid sequence. The phrase nucleotide sequence that encodes a protein oran RNA may also include introns to the extent that the nucleotidesequence encoding the protein may in some version contain an intron(s).

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo,ex vivo or in vitro.

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner. A “constitutive” promoteris a nucleotide sequence which, when operably linked with apolynucleotide which encodes or specifies a gene product, causes thegene product to be produced in a cell under most or all physiologicalconditions of the cell.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In some embodiments, the patient, subject or individual is a human.Examples of subjects include humans, dogs, cats, mice, rats, andtransgenic species thereof. In some embodiments, the subject is anon-human mammal. In some embodiments, the subject is a non-humanprimate. In some embodiments, the subject is a rodent. In someembodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog.In some embodiments, the subject is a vertebrate, an amphibian, areptile, a fish, an insect, a fly, or a nematode. In some embodiments,the subject is a research animal. In some embodiments, the subject isgenetically engineered, e.g., a genetically engineered non-humansubject. The subject may be of either sex and at any stage ofdevelopment. In some embodiments, the subject has a NET-mediated orNET-associated condition or disease (e.g., ARDS). In other embodiments,the subject is a healthy volunteer.

By “NETs” is meant Neutrophil Extracellular Traps, which areextracellular webs of chromatin, i.e., DNA and associated nuclearproteins, released by neutrophils in a controlled process called, e.g.,NETosis.

Where used with respect to pseudouridine, the terms “substituted” or“substitution” refer to an RNA wherein one or more pseudouridinenucleotides occupy sequence position(s) that are otherwise described asoccupied, or otherwise would be occupied, by uridine(s) in one or morenucleic acid sequences or embodiments described or referenced herein,including those uridine(s) implied to occur in RNAs that arecomplementary to any DNA sequence described or referenced herein. Forexample, of the sum of uridine and pseudouridine nucleotides in aparticular nucleic acid, pseudouridine can account for at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 97%, at least 99%, at least 99.5%, or at least 99.9%. In someembodiments where substantially all positions that would otherwise beoccupied by uridines are occupied by pseudouridines, at least 90%, atleast 95%, at least 97%, at least 99%, at least 99.5%, or at least 99.9%of such positions are occupied by pseudouridines.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

As used herein, the terms “treatment,” “treat,” and “treating” refer toa clinical intervention aimed to reverse, alleviate, delay the onset of,or inhibit the progress of a disease or disorder, or one or moresymptoms thereof, as described herein. In some embodiments, treatmentmay be administered after one or more symptoms have developed and/orafter a disease has been diagnosed. In other embodiments, treatment maybe administered in the absence of symptoms, e.g., to prevent or delayonset of a symptom or inhibit onset or progression of a disease. Forexample, treatment may be administered to a susceptible individual priorto the onset of symptoms (e.g., in light of a history of symptoms and/orin light of genetic or other susceptibility factors). Treatment may alsobe continued after symptoms have resolved, for example, to prevent ordelay their recurrence.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Sequences

The following amino acid (AA) or nucleotide (nt) sequences arereferenced herein:

SEQ ID NO: 1 (human DNASE1; AA sequence)MRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK SEQ ID NO: 2 (human DNASE1L3; AA sequence)MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRSSEQ ID NO: 3 (human DNASE1-T2A-human DNASE1L3; AA sequence)MRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLKEGRGSLLTCGDVEENPGPMSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSWPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS SEQ ID NO: 4 (transmembrane IL15; AA sequence)MALPVTALLLPLALLLHAARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNGGSDYKDDDDKSEQ ID NO: 5 (IL15-TGFbeta fusion protein; AA sequence)MGWSCIILFLVATATGVHSDYKDDDDKTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGTGGSSGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSEQ ID NO: 6 (IL15-TGFbeta fusion protein, alternate; AA sequence)MGWSCIILFLVATATGVHSDYKDDDDKNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGTGGSSGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSEQ ID NO: 7 (Influenza NS1; AA sequence)MDSNTVSSFQVDCFLWHVRKQVADQELGDAPFLDRLRRDQKSLKGRGSTLGLNIETATCVGKQIVERILKEESDEAFRMTMASALASRYLTDMTIEEMSRDWFMLMPKQKVAGPLCVRMDQAIMDKNIILKANFSVIFDRLETLTLLRAFTEEGAIVGEISPLPSLPGHTNEDVKNAIGVLIGGLEWNDNTVRVSETLQRFAWRSSNENGGPPLTPTQKRKMAGKIRSEVSEQ ID NO: 8 (Vaccinia E3; AA sequence)MSKIYIDERSDAEIVCAAIKNIGIEGATAAQLTRQLNMEKREVNKALYDLQRSAMVYSSDDIPPRWFMTTEADKPDADAMADVIIDDVSREKSMREDHKSFDDVIPAKKIIDWKDANPVTIINEYCQITKRDWSFRIESVGPSNSPTFYACVDIDGRVFDKADGKSKRDAKNNAAKLAVDKLLGYVIIRFSEQ ID NO: 9 (Human XBP1-iso2; AA sequence)MVVVAAAPNPADGTPKVLLLSGQPASAAGAPAGQALPLMVPAQRGASPEAASGGLPQARKRQRLTHLSPEEKALRRKLKNRVAAQTARDRKKARMSELEQQVVDLEEENQKLLLENQLLREKTHGLVVENQELRQRLGMDALVAEEEAEAKGNEVRPVAGSAESAAGAGPVVTPPEHLPMDSGGIDSSDSESDILLGILDNLDPVMFFKCPSPEPASLEELPEVYPEGPSSLPASLSLSVGTSSAKLEAINELIRFDHIYTKPLVLEIPSETESQANVVVKIEEAPLSPSENDHPEFIVSVKEEPVEDDLVPELGISNLLSSSHCPKPSSCLLDAYSDCGYGGSLSPFSDMSSLLGVNHSWEDTFANELFPQLISVSEQ ID NO: 10 (DP71L; AA sequence)MGGRRRKKRTNDTKHVRFAAAVEVWEADDIERKGPWEQVAVDRFRFQRRIASVEELLSTVLLRQK KLLEQQSEQ ID NO: 11 (PP1-GADD34 eIF2a binding protein; AA sequence)MSDSEKLNLDSIIGRLLEVQGSRPGKNVQLTENEIRGLCLKSREIFLSQPILLELEAPLKICGDIHGQYYDLLRLFEYGGFPPESNYLFLGDYVDRGKQSLETICLLLAYKIKYPENFFLLRGNHECASINRIYGFYDECKRRYNIKLWKTFTDCFNCLPIAAIVDEKIFCCHGGLSPDLQSMEQIRRIMRPTDVPDQGLLCDLLWSDPDKDVQGWGENDRGVSFTFGAEVVAKFLHKHDDLICRAHQVVEDGYEFFAKRQLVTLFSAPNYCGEFDNAGAMMSVDETLMCSFQILKPADKNKGKYGQFSGLNPGGRPITPPRNSAKAKKARQGPWEQLARDRSREARRITQAQEELSPCLTPAARARAWASEQ ID NO: 12 (B18R; AA sequence)MGTMKMMVHIYFVSLLLLLFHSYAIDIENEITEFFNKMRDTLPAKDSKWLNPACMFGGTMNDIAALGEPFSAKCPPIEDSLLSHRYKDYVVKWERLEKNRRRQVSNKRVKHGDLWIANYTSKFSNRRYLCTVTTKNGDCVQGIVRSHIRKPPSCIPKTYELGTHDKYGIDLYCGILYAKHYNNITWYKDNKEINIDDIKYSQTGKELIIHNPELEDSGRYDCYVHYDDVRIKNDIVVSRCKILTVIPSQDHRFKLILDPKINVTIGEPANITCTAVSTSLLIDDVLIEWENPSGWLIGFDFDVYSVLTSRGGITEATLYFENVTEEYIGNTYKCRGHNYYFEKTLTTTVVLESEQ ID NO: 13 (IRES; Internal Ribosomal Entry Site; nt sequence)GcggccatcgatgtcgacaactaacttaagctagcaacggtttccctctagcgggatcaattccgccccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgataataccaattcgccgccaccSEQ ID NO: 14 (T2A; AA sequence) EGRGSLLTCGDVEENPGPSEQ ID NO: 15 (5′UTR of pSFVC vector; nt sequence)atggcggatgtgtgacatacacgacgccaaaagattttgttccagctcctgccacctccgctacgcgagagattaaccacccacg SEQ ID NO: 16 (3′UTR of pSFVC vector; nt sequence)taataaccgggcaggggggatcccgggtaattaattgaattacatccctacgcaaacgttttacggccgccggtggcgcccgcgcccggcggcccgtccctggccgttgcaggccactccggtggctcccgtcgtccccgacttccaggcccagcagatgcagcaactcatcagcgccgtaaatgcgctgacaatgagacagaacgcaattgctcctgctaggcctcccaaaccaaagaagaagaagacaaccaaaccaaagccgaaaacgcagcccaagaagatcaacggaaaaacgcagcagcaaaagaagaaagacaagcaagccgacaagaagaagaagaaacccggaaaaagagaaagaatgtgcatgaagattgaaaatgactgtatctatgcggctagccacagtaacgtagtgtttccagacatgtcgggcaccgcactatcatgggtgcagaaaatctcgggtggtctgggggccttcgcaatcggcgctatcctggtgctggttgtggtcacttgcattgggctccgcagataagttagggtaggcaatggcattgatatagcaagaaaattgaaaacagaaaaagttagggtaagcaatggcatataaccataactgtataacttgtaacaaagcgcaacaagacctgcgcaattggccccgtggtccgcctcacggaaactcggggcaactcatattgacacattaattggcaataattggaagcttacataagcttaattcgacgaataattggatttttattttattttgcaattggtttttaatatttcc SEQ ID NO: 17 (NSP1-NSP4; AA sequence)MAAKVHVDIEADSPFIKSLQKAFPSFEVESLQVTPNDHANARAFSHLATKLIEQETDKDTLILDIGSAPSRRMMSTHKYHCVCPMRSAEDPERLVCYAKKLAAASGKVLDREIAGKITDLQTVMATPDAESPTFCLHTDVTCRTAAEVAVYQDVYAVHAPTSLYHQAMKGVRTAYWIGFDTTPFMFDALAGAYPTYATNWADEQVLQARNIGLCAASLTEGRLGKLSILRKKQLKPCDTVMFSVGSTLYTESRKLLRSWHLPSVFHLKGKQSFTCRCDTIVSCEGYVVKKITMCPGLYGKTVGYAVTYHAEGFLVCKTTDTVKGERVSFPVCTYVPSTICDQMTGILATDVTPEDAQKLLVGLNQRIVVNGRTQRNTNTMKNYLLPIVAVAFSKWAREYKADLDDEKPLGVRERSLTCCCLWAFKTRKMHTMYKKPDTQTIVKVPSEFNSFVIPSLWSTGLAIPVRSRIKMLLAKKTKRELIPVLDASSARDAEQEEKERLEAELTREALPPLVPIAPAETGVVDVDVEELEYHAGAGVVETPRSALKVTAQPNDVLLGNYVVLSPQTVLKSSKLAPVHPLAEQVKIITHNGRAGRYQVDGYDGRVLLPCGSAIPVPEFQALSESATMVYNEREFVNRKLYHIAVHGPSLNTDEENYEKVRAERTDAEYVFDVDKKCCVKREEASGLVLVGELTNPPFHEFAYEGLKIRPSAPYKTTVVGVFGVPGSGKSAIIKSLVTKHDLVTSGKKENCQEIVNDVKKHRGLDIQAKTVDSILLNGCRRAVDILYVDEAFACHSGTLLALIALVKPRSKVVLCGDPKQCGFFNMMQLKVNFNHNICTEVCHKSISRRCTRPVTAIVSTLHYGGKMRTTNPCNKPIIIDTTGQTKPKPGDIVLTCFRGWVKQLQLDYRGHEVMTAAASQGLTRKGVYAVRQKVNENPLYAPASEHVNVLLTRTEDRLVWKTLAGDPWIKVLSNIPQGNFTATLEEWQEEHDKIMKVIEGPAAPVDAFQNKANVCWAKSLVPVLDTAGIRLTAEEWSTIITAFKEDRAYSPVVALNEICTKYYGVDLDSGLFSAPKVSLYYENNHWDNRPGGRMYGFNAATAARLEARHTFLKGQWHTGKQAVIAERKIQPLSVLDNVIPINRRLPHALVAEYKTVKGSRVEWLVNKVRGYHVLLVSEYNLALPRRRVTWLSPLNVTGADRCYDLSLGLPADAGRFDLVFVNIHTEFRIHHYQQCVDHAMKLQMLGGDALRLLKPGGSLLMRAYGYADKISEAVVSSLSRKFSSARVLRPDCVTSNTEVFLLFSNFDNGKRPSTLHQMNTKLSAVYAGEAMHTAGCAPSYRVKRADIATCTEAAVVNAANARGTVGDGVCRAVAKKWPSAFKGEATPVGTIKTVMCGSYPVIHAVAPNFSATTEAEGDRELAAVYRAVAAEVNRLSLSSVAIPLLSTGVFSGGRDRLQQSLNHLFTAMDATDADVTIYCRDKSWEKKIQEAIDMRTAVELLNDDVELTTDLVRVHPDSSLVGRKGYSTTDGSLYSYFEGTKFNQAAIDMAEILTLWPRLQEANEQICLYALGETMDNIRSKCPVNDSDSSTPPRTVPCLCRYAMTAERIARLRSHQVKSMVVCSSFPLPKYHVDGVQKVKCEKVLLFDPTVPSVVSPRKYAASTTDHSDRSLRGFDLDWTTDSSSTASDTMSLPSLQSCDIDSIYEPMAPIVVTADVHPEPAGIADLAADVHPEPADHVDLENPIPPPRPKRAAYLASRAAERPVPAPRKPTPAPRTAFRNKLPLTFGDFDEHEVDALASGITFGDFDDVLRLGRAGAYIFSSDTGSGHLQQKSVRQHNLQCAQLDAVEEEKMYPPKLDTEREKLLLLKMQMHPSEANKSRYQSRKVENMKATVVDRLTSGARLYTGADVGRIPTYAVRYPRPVYSPTVIERFSSPDVAIAACNEYLSRNYPTVASYQITDEYDAYLDMVDGSDSCLDRATFCPAKLRCYPKHHAYHQPTVRSAVPSPFQNTLQNVLAAATKRNCNVTQMRELPTMDSAVFNVECFKRYACSGEYWEEYAKQPIRITTENITTYVTKLKGPKAAALFAKTHNLVPLQEVPMDRFTVDMKRDVKVTPGTKHTEERPKVQVIQAAEPLATAYLCGIHRELVRRLNAVLRPNVHTLFDMSAEDFDAIIASHFHPGDPVLETDIASFDKSQDDSLALTGLMILEDLGVDQYLLDLIEAAFGEISSCHLPTGTRFKFGAMMKSGMFLTLFINTVLNITIASRVLEQRLTDSACAAFIGDDNIVHGVISDKLMAERCASWVNMEVKIIDAVMGEKPPYFCGGFIVFDSVTQTACRVSDPLKRLFKLGKPLTAEDKQDEDRRRALSDEVSKWFRTGLGAELEVALTSRYEVEGCKSILIAMATLARDIKAFKKLRGPVIHLYGGPRLVR SEQ ID NO: 18 (26S Promoter; nt sequence)tacacagaattctgattatagcgcactattatagcaccatgaattacatccctacgcaaacgttttacggccgccggtggcgcccgcgcccggcggcccgtccctggccgttgcaggccactccgSEQ ID NO: 19 (C Protein fragment; AA sequence)VAPVVPDFQAQQMQQLISAVNALTMRQNAIAPARPPKPKKKKTTKPKPKTQPKKINGKTQQQKKKDKQADKKKKKPGKRERMCMKIENDCIFEVKHEGKVTGYACLVGDKVMKPAHVKGVIDNADLAKLAFKKSSKYDLECAQIPVHMRSDASKYTHEKPEGHYNWHHGAVQYSGGRFTIPTGAGKPGDSGRPIFDNKGRVVAIVLGGANEGSRTALSVVTWNKDMVTRVTPEGSEEWDPSEQ ID NO: 20 (Kozak Sequence; nt sequence) gccgccaccatgaSEQ ID NO: 21 (anti-BCMA, anti-CD3 bispecific T-cell engager,with signal sequence)MYRMQLLSCIALSLALVTNSDIVLTQSPASLAVSPGQRATITCRASESVSFLGINLIHWYQQKPGQPPKLLIYSASNLQSGVPARFSGSGSGTDFTLTISSVEPEDTANYYCLQSRTLPRTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGGSVKISCKASGYTFTSYSINWVRQAPGKGLEWVGWINTETREPAYAQGFTGRFTFSADTSKSMAYLQINSLRAEDTAVYYCALDYLYSLDFWGQGTLVTVSSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSKGYTNYNQKFKGRVTMTADKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERATLSCRASSSVSYLNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTDFTLTISSLQAEDEATYYCQQWSSNPLTFGQGTKLEIKSEQ ID NO: 22 (anti-BCMA, anti-CD3 bispecific T-cell engager,without signal sequence)DIVLTQSPASLAVSPGQRATITCRASESVSFLGINLIHWYQQKPGQPPKLLIYSASNLQSGVPARFSGSGSGTDFTLTISSVEPEDTANYYCLQSRTLPRTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGGSVKISCKASGYTFTSYSINWVRQAPGKGLEWVGWINTETREPAYAQGFTGRFTFSADTSKSMAYLQINSLRAEDTAVYYCALDYLYSLDFWGQGTLVTVSSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSKGYTNYNQKFKGRVTMTADKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERATLSCRASSSVSYLNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTDFTLTISSLQAEDEATYYCQQWSSNPLTFGQGTKLEIKSEQ ID NO: 23 (5′UTR for Inventive Construct; nt sequence)AGACCCAAGCTGGCTAGCtctaaagaagcccctgggagcacagctcatcaccSEQ ID NO: 24 (3′UTR for Inventive Construct; nt sequence)ctatgaagaaggaaggcatccagaccagaaaccgaaaaatgtctagcaaatccaaaaagtgcaaaaaagtgcatgactcactggaggacttccccaagaacagctcgtttaacccggccgccctctccagacacatgtcctccctgagccacatctcgcccttcagccactccagccacatgctgaccacgcccacgccgatgcacccgccatccagc

Where a polynucleotide sequence or “open reading frame” is described as,or by reference to, an amino acid sequence, the skilled person willunderstand that a corresponding polynucleotide sequence can be readilyobtained that encodes the amino acid sequence. Generally, naturallyoccurring polynucleotide sequences (or fragments thereof) that encode anaturally occurring protein of interest (or fragment thereof) can beobtained from public sequence databases.

Exemplary Nucleotide Constructs

Provided herein are examples of RNA constructs useful to modify a cellto secrete one or more DNAse(s) in accordance with the invention:

Construct A:

Cap 5′ UTR NSP1-NSP4 26S promoter DNASE1 3′ UTR polyAwherein “Cap” refers to a 5′ cap, e.g., a 5-methylguanosine cap or othercap known in the art; “5′ UTR” refers to a 5′ untranslated region, e.g.,the sequence of SEQ ID NO: 15; “NSP1-NSP4” refers to the nonstructuralproteins of alphavirus NSP1, NSP2, NSP3, and NSP4, which can beexemplified as a combined unit, e.g., by the sequence of SEQ ID NO: 17;“26S promoter” is a transcriptional promoter exemplified by the sequenceof SEQ ID NO: 18; DNASE1 encodes a DNASE1 protein, e.g., of the sequenceof SEQ ID NO: 1 and in some embodiments preferably starts with a Kozakconsensus sequence; “3′ UTR” refers to a 3′ untranslated region, e.g.,the sequence of SEQ ID NO: 16; and “polyA” refers to a polyadenine tail,e.g., of 70 adenine units. The region denoted as “DNASE1” preferablyincludes a Kozak consensus sequence.

Construct B:

Cap 5′ UTR NSP1-NSP4 26S promoter DNASE1L3 3′ UTR polyAwherein terms are as defined for Construct A, and wherein DNASE1L3encodes a DNASE1L3 protein, e.g., of the sequence of SEQ ID NO: 2 and insome embodiments preferably starts with a Kozak consensus sequence.

Construct C:

Cap 5′ UTR NSP1-NSP4 26S promoter DNASE1 T2A DNASE1L3 3′ UTR polyAwherein terms are as defined for Constructs A and B, and wherein “T2A”refers to a self-cleaving peptide, e.g., of the sequence of SEQ ID NO:14.

Construct D:

Cap 5′ UTR NSP1-NSP4 26S promoter DNASE1 T2A DNASE1L3 3′ UTR polyAwherein terms are as defined for Construct D.

Construct E:

Cap 5′ UTR NSP1-NSP4 26S promoter DNASE1 IRES DNASE1L3 3′ UTR polyAwherein terms are as defined for Construct D, and wherein “IRES” refersto an Internal Ribosome Entry Site, e.g., of the sequence of SEQ ID NO:13.

Construct F:

Cap 5′ UTR NSP1-NSP4 26S promoter C protein DNASE1 T2A DNASE1L3 3′ UTRpolyAwherein terms are as defined for Construct D, and wherein “C protein”refers to a fragment of a viral C protein, e.g., of the sequence of SEQID NO: 19. Alternatively, the fragment of a viral C protein can consistof the first 34 or more amino acids recited in SEQ ID NO: 19.

Construct G:

Cap 5′ UTR NSP1-NSP4 26S promoter C protein DNASE1L3 T2A DNASE1 3′ UTRpolyAwherein terms are as defined for Construct F.

Construct H:

Cap 5′ UTR NSP1-NSP4 26S promoter C protein DNASE1 IRES DNASE1L3 3′ UTRpolyAwherein terms are as defined for Construct E and Construct F.

Construct I:

Cap 5′ UTR NSP1-NSP4 26S promoter C protein DNASE1L3 T2A DNASE1 T2ABispec 3′ UTR polyAwherein terms are as defined for Construct G, and wherein “Bispec”refers to a bispecific antibody, e.g., an anti-BCMA, anti-CD3 bispecificantibody, as further described below.

The above embodiments are particularly well suited for the modificationof cells by introduction of self-amplifying RNA (saRNA).

Novel Cells Modified to Secrete One or More DNAse Enzymes

In one aspect, the invention provides a cell modified to secrete a DNaseenzyme. In some embodiments, the cell is a mammalian cell. In someembodiments, the cell is a white blood cell, e.g., an NK cell, T cell,CD8+ cell, CD4+ cell, monocyte, macrophage, or CD34+ cell. In someembodiments, the cell is a Mesenchymal Stem (or Stromal) Cell (MSC). Insome embodiments, the cell is a stem cell. In some embodiments, the cellis engineered to secrete DNAse.

In some embodiments, the DNAse is DNASE1. In some embodiments, the DNAseis DNASE1L1. In some embodiments, the DNAse is DNASE1L2. In someembodiments, the DNAse is DNASE1L3. In some embodiments, the DNAse isDNASE2A. In some embodiments, the DNAse is DNASE2B. In some embodiments,the DNAse is L-DNASEII. In some embodiments, the DNAse is CAD, alsoknown as DFF40 or DFFB. In some embodiments, the DNAse is EndoG, alsoknown as Endonuclease G.

In some embodiments, the cell is modified to secrete two or more DNaseenzymes selected from the group consisting of: DNASE1, DNASE1L1,DNASE1L2, DNASE1L3, DNASE2A, DNAS2B, L-DNASEII, CAD, and EndoG. In someembodiments, the cell is modified to secrete: DNASE1 and DNASE1L1;DNASE1 and DNASE1L2; DNASE1 and DNASE1L3; DNASE1 and DNASE2A; DNASE1 andDNASE2B; DNASE1 and L-DNASEII; DNASE1 and CAD; DNASE1 and EndoG;DNASE1L1 and DNASE1L2; DNASE1L1 and DNASE1L3; DNASE1L1 and DNASE2A;DNASE1L1 and DNASE2B; DNASE1L1 and L-DNASEII; DNASE1L1 and CAD; DNASE1L1and EndoG; DNASE1 and DNASE1L3; DNASE1 and DNASE2A; DNASE1 and DNASE2B;DNASE1 and L-DNASEII; DNASE1 and CAD; DNASE1 and EndoG; DNASE1L2 andDNASE1L3; DNASE1L2 and DNASE2A; DNASE1L2 and DNASE2B; DNASE1L2 andL-DNASEII; DNASE1L2 and CAD; DNASE1L2 and EndoG; DNASE1L3 and DNASE2A;DNASE1L3 and DNASE2B; DNASE1L3 and L-DNASEII; DNASE1L3 and CAD; DNASE1L3and EndoG; DNASE2A and DNASE2B; DNASE2A and L-DNASEII; DNASE2A and CAD;DNASE2A and EndoG; DNASE2B and L-DNASEII; DNASE2B and CAD; DNASE2B andEndoG; L-DNASEII and CAD; L-DNASEII and EndoG; or CAD and EndoG.

In some embodiments, the cell is modified to express a DNAse comprisingthe sequence of SEQ ID NO: 1. In some embodiments, the cell is modifiedto secrete a protein having the sequence of SEQ ID NO: 1. In someembodiments, the cell is modified to express a DNAse comprising thesequence of SEQ ID NO: 2. In some embodiments, the cell is modified tosecrete a protein having the sequence of SEQ ID NO: 2. In someembodiments, the cell is modified to express a protein comprising thesequence of SEQ ID NO: 3. In some embodiments, the cell is modified tosecrete a protein having the sequence of SEQ ID NO: 3. In someembodiments, the cell is modified to express a first DNAse comprisingthe sequence of SEQ ID NO: 1 and a second DNAse comprising the sequenceof SEQ ID NO: 2. In some embodiments, the cell is modified to secrete afirst DNAse comprising the sequence of SEQ ID NO: 1 and a second DNAsecomprising the sequence of SEQ ID NO: 2.

In some embodiments, the cell is modified to express a DNAse comprisinga sequence having at least 90% sequence identity to the entire sequenceof SEQ ID NO: 1. In some embodiments, the cell is modified to secrete aprotein comprising a sequence having at least 90% sequence identity tothe entire sequence of SEQ ID NO: 1. In some embodiments, the cell ismodified to express a DNAse comprising a sequence having at least 90%sequence identity to the entire sequence of SEQ ID NO: 2. In someembodiments, the cell is modified to secrete a protein having at least90% sequence identity to the entire sequence of SEQ ID NO: 2. In someembodiments, the cell is modified to express a protein comprising asequence having at least 90% sequence identity to the entire sequence ofSEQ ID NO: 3. In some embodiments, the cell is modified to secrete aprotein comprising a sequence having at least 90% sequence identity tothe entire sequence of SEQ ID NO: 3. In some embodiments, the cell ismodified to express a first DNAse comprising a sequence having at least90% sequence identity to the entire sequence of SEQ ID NO: 1 and asecond DNAse comprising a sequence having at least 90% sequence identityto the entire sequence of SEQ ID NO: 2. In some embodiments, the cell ismodified to secrete a first DNAse comprising a sequence having at least90% sequence identity to the entire sequence of SEQ ID NO: 1 and asecond DNAse comprising a sequence having at least 90% sequence identityto the entire sequence of SEQ ID NO: 2.

In some embodiments, the cell is modified to express a DNAse comprisinga sequence having at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least99.5% identical to the entire sequence of SEQ ID NO: 1. In someembodiments, the cell is modified to secrete a protein comprising asequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, or at least99.5% identical to the entire sequence of SEQ ID NO: 1. In someembodiments, the cell is modified to express a DNAse comprising asequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, or at least99.5% identical to the entire sequence of SEQ ID NO: 2. In someembodiments, the cell is modified to secrete a protein having at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or at least 99.5% identical to theentire sequence of SEQ ID NO: 2. In some embodiments, the cell ismodified to express a protein comprising a sequence having at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or at least 99.5% identical to the entiresequence of SEQ ID NO: 3. In some embodiments, the cell is modified tosecrete a protein comprising a sequence having at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or at least 99.5% identical to the entire sequence ofSEQ ID NO: 3. In some embodiments, the cell is modified to express afirst DNAse comprising a sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or at least 99.5% identical to the entire sequence of SEQ IDNO: 1 and a second DNAse comprising a sequence having at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or at least 99.5% identical to the entiresequence of SEQ ID NO: 2. In some embodiments, the cell is modified tosecrete a first DNAse comprising a sequence having at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or at least 99.5% identical to the entiresequence of SEQ ID NO: 1 and a second DNAse comprising a sequence havingat least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or at least 99.5% identical tothe entire sequence of SEQ ID NO: 2.

In some embodiments, the cell is modified by introduction of one of morepolynucleotide constructs selected from the group consisting ofConstruct A, Construct B, Construct C, Construct D, Construct E,Construct F, Construct G, Construct H, and Construct I. In someembodiments, the cell is modified by introduction of a polynucleotide ofConstruct A. In some embodiments, the cell is modified by introductionof a polynucleotide of Construct B. In some embodiments, the cell ismodified by introduction of a polynucleotide of Construct C. In someembodiments, the cell is modified by introduction of a polynucleotide ofConstruct D. In some embodiments, the cell is modified by introductionof a polynucleotide of Construct E. In some embodiments, the cell ismodified by introduction of a polynucleotide of Construct F. In someembodiments, the cell is modified by introduction of a polynucleotide ofConstruct G. In some embodiments, the cell is modified by introductionof a polynucleotide of Construct H. In some embodiments, the cell ismodified by introduction of a polynucleotide of Construct I.

In some of the foregoing embodiments, the cell is modified to secreteone or more DNase enzymes that the cell does not normally express, ordoes not normally secrete. In some of the foregoing embodiments, thecell is modified to overexpress one or more DNase enzymes. In some ofthe foregoing embodiments, the cell is modified to secrete one or moreDNase enzymes that it normally expresses but does not normally secrete.

In some embodiments, the modified cells of the invention are modifiedthrough the introduction of DNA into the cells; in some suchembodiments, the DNA that comprises a sequence that is complementary toone or more RNA sequences described herein, or a portion thereof. Insome embodiments, the modified cells of the invention are modifiedthrough the introduction of RNA into the cells (e.g., an RNA comprisinga sequence that encodes one or more DNAse enzymes, e.g., DNASE1 and/orDNASE1L3, as described herein). In some embodiments, the RNA is amessenger RNA (mRNA). In some embodiments, the RNA is a self-amplifyingRNA (saRNA). In some embodiments, the RNA comprises pseudouridine. Insome embodiments, the mRNA is artificially enriched in pseudouridine. Insome embodiments, substantially all of the uridine nucleotides of theRNA are substituted with pseudouridine. Methods for incorporatingpseudouridine into an RNA are generally known in the art.

In some embodiments, two or more mRNA molecules encoding differentproteins are used to modify the cells. In some embodiments, two or moresaRNA molecules encoding different proteins are used to modify thecells. In some embodiments, two or more protein products are encoded onthe same RNA molecule used to modify the cells. In some embodiments, twoor more protein products are encoded on the same mRNA molecule used tomodify the cells. In some embodiments, two or more protein products areencoded on the same saRNA molecule used to modify the cells. In someembodiments, two or more DNAse enzymes, e.g., DNASE1 and DNASE1L3, areencoded on the same RNA molecule used to modify the cells. In someembodiments, two or more DNAse enzymes, e.g., DNASE1 and DNASE1L3, areencoded on the same mRNA molecule used to modify the cells. In someembodiments, two or more DNAse enzymes, e.g., DNASE1 and DNASE1L3, areencoded on the same saRNA molecule used to modify the cells.

In some embodiments, two separate mRNA molecules encoding DNASE1 andDNASE1L3, respectively, are introduced into cell(s) to cause the cell(s)to express and/or secrete each of DNASE1 and DNASE1L3. In someembodiments, both of the mRNA molecules are artificially enriched inpseudouridine. In some embodiments, substantially all of the uridinenucleotides of the mRNA molecules are substituted with pseudouridine. Insome embodiments, the cell is modified by introduction of aDNASE1-encoding mRNA that is enriched in pseudouridine and aDNASE1L3-encoding mRNA that is not enriched in pseudouridine. In someembodiments, the cell is modified by introduction of a DNASE1-encodingmRNA that is not enriched in pseudouridine and a DNASE1L3-encoding mRNAthat is enriched in pseudouridine. In some embodiments, the cell ismodified by introduction of a DNASE1-encoding mRNA wherein substantiallyall of the uridine nucleotides are substituted with pseudouridine and aDNASE1L3-encoding mRNA that is not enriched in pseudouridine.

In some embodiments, a cell is modified by introduction of aDNASE1-encoding mRNA that is not enriched in pseudouridine and aDNASE1L3-encoding mRNA wherein substantially all of the uridinenucleotides are substituted with pseudouridine.

In some embodiments wherein a cell is modified to express DNAseenzyme(s) encoded by an saRNA, the same cell is also modified to expressan RNA-dependent RNA polymerase (RDRP), whereby the cell amplifies,i.e., replicates, the saRNA(s) introduced into it. In some embodiments,the saRNA molecule encoding the RDRP protein is separate from the saRNAmolecule encoding one or more DNAse proteins.

In some embodiments, an in vitro transcribed RNA can be introduced to acell as a form of transfection. The RNA is produced by in vitrotranscription using a polymerase chain reaction (PCR)-generatedtemplate. In some embodiments, the RNA is transcribed directly from alinearized plasmid. DNA of interest from any source can be directlyconverted by PCR into a template for in vitro mRNA synthesis usingappropriate primers and RNA polymerase. The source of the DNA can be,for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNAsequence, or any other appropriate source of DNA. The desired templatefor in vitro transcription is the DNAse of the present invention.

Nucleic acid, including but not limited to RNA, can be introduced intotarget cells using any of a number of different methods, for instance,commercially available methods which include, but are not limited to,electroporation (4D NUCLEOFECTOR® or AMAXA NUCLEOFECTOR-II® (Lonza,Basel, Switzerland), MaxCyte apparatuses (MaxCyte, Gaithersburg, Md.),ECM 830 BTX (Harvard Instruments, Boston, Mass.), GENE PULSER II®(BioRad, Denver, Colo.), or MULTIPORATOR® (Eppendorf, Hamburg Germany)),flow electroporation (e.g., U.S. Pat. Pub. No. 2017/0218355) mechanicalmembrane disruption (e.g., cell squeezing, see U.S. Pat. Pub. No.2014/287509A1), cationic liposome mediated transfection usinglipofection, nanoparticle-mediated delivery (e.g., with lipidencapsulated nanoparticles, gold, or polymer encapsulatednanoparticles), polymer encapsulation, peptide mediated transfection, orbiolistic particle delivery systems, such as “gene guns” (see, e.g.,Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).

In some embodiments, the DNAse sequence(s) (e.g., nucleic acidsequence(s) encoding a DNAse, e.g., DNASE1 and/or DNASE1L3, as describedherein) are delivered into cells (e.g., NK cells, T cells, monocytes,macrophages, MSCs, or stem cells) using a retroviral or lentiviralvector. DNAse-expressing retroviral and lentiviral vectors can bedelivered into different types of eukaryotic cells as well as intotissues and whole organisms using transduced cells as carriers orcell-free local or systemic delivery of encapsulated, bound or nakedvectors. The method used can be for any purpose where stable expressionis required or sufficient.

In some embodiments, one or more DNAses (e.g., DNASE1 and/or DNASE1L3)can be expressed in the cells (e.g., NK cells, T cells, monocytes,macrophages, MSCs, or stem cells) by way of transposons and/orretrotransposons, e.g., piggyBac™ transposon system (System Biosiences,Palo Alto, Calif.), and sleeping beauty transposon system (see, e.g.,Geurts et al., Mol. Ther. 2003; 8:108-117).

In some embodiments, the cell that is modified to express and/or secreteone or more DNAses ((e.g., DNASE1 and/or DNASE1L3) is further modifiedto express and/or secrete one or more survival factors, e.g.,transmembrane IL-15 (e.g., SEQ ID NO:4), secreted IL-15, IL15-TGFβfusion protein (e.g., SEQ ID NO:6), and/or IL-15 TGFβ fusion protein(e.g., SEQ ID NO: 7).

In some embodiments, the cell that is modified to express and/or secreteone or more DNAses ((e.g., DNASE1 and/or DNASE1L3) is further modifiedto express one or more translation enhancers, e.g., influenza NS1 (e.g.,SEQ ID NO: 7), Vaccinia E3 (e.g., SEQ ID NO: 8), human XBP1-iso2 (e.g.,SEQ ID NO: 9), DP71L (e.g., SEQ ID NO: 10), PP1-GADD34 eIF2a bindingprotein (e.g., SEQ ID NO: 11), and/or B 18R (e.g., SEQ ID NO: 12).

In any of the above embodiments wherein a nucleic acid or nucleic acidconstruct is introduced into a cell, that nucleic acid or nucleic acidconstruct can be an exogenous nucleic acid or exogenous nucleic acidconstruct. Generally, the exogenous nucleic acid or nucleic acidconstruct can be any nucleic acid or nucleic acid construct disclosedherein. In some embodiments, an exogenous RNA or RNA construct isintroduced into a cell to modify the cell. In some embodiments, anexogenous DNA or DNA construct is introduced into a cell to modify thecell.

Sources of Homospecific Cells

Prior to modification of the cells of the invention, includinghomospecific cells, for example NK cells, T cells, monocytes,macrophages, MSCs, stem cells, a source of cells is obtained from asubject of the same species. In some embodiments, the cells are derivedfrom a subject different than the intended recipient of the modifiedcells (i.e., heterologous use, including clinical heterologous use). Insome other embodiments, the cells are derived from the same subject forwhom they are intended once modified (i.e., autologous use).Homospecific cells can be obtained from a number of sources, includingperipheral blood mononuclear cells, bone marrow, adipose tissue, lymphnode tissue, cord blood, thymus tissue, tissue from a site of infection,ascites, pleural effusion, spleen tissue, blod, and tumors. Homospecificcells, including stem cells, may be generated from induced pluripotentstem cells or hematopoietic stem cells or progenitor cells. In someembodiments of the present invention, any number of cell lines,including but not limited to NK cell lines, T cell lines, and/or stemcell lines, available in the art, may be used. In some embodiments ofthe present invention, homospecific blood cells (e.g., NK cells, Tcells, monocytes, macrophages, MSCs, stem cells) can be obtained from aunit of blood collected from a subject using any number of techniquesknown to the skilled artisan, such as Ficoll™ separation. In someembodiments, cells from the circulating blood of an individual areobtained by apheresis. The apheresis product typically containslymphocytes, including T cells, monocytes, granulocytes, B cells, NKcells, other nucleated white blood cells including CD34+ cells, redblood cells, and platelets. In some embodiments, the cells collected byapheresis may be washed to remove the plasma fraction and to place thecells in an appropriate buffer or media for subsequent processing steps.In some embodiments of the invention, the cells are washed withphosphate buffered saline (PBS) or other suitable fluid. After washing,the cells may be resuspended in a variety of biocompatible buffers, suchas, for example, Ca²⁺-free, Mg²⁺-free PBS, PlasmaLyte A, or other salinesolution with or without buffer. Alternatively, the undesirablecomponents of the apheresis sample may be removed and the cells directlyresuspended in culture media.

In another embodiment, blood cells, including homospecific blood cells,for example, NK cells, T cells, monocytes, macrophages, or CD34+ cellsare isolated from peripheral blood lymphocytes by lysing the red bloodcells and depleting the monocytes, for example, by centrifugationthrough a PERCOLL™ gradient or by counterflow centrifugal elutriation. Aspecific subpopulation of cells, such as NK cells, T cells, or CD34+cells, can be further isolated by positive or negative selectiontechniques.

In some embodiments, MSCs are isolated from: bone marrow mononuclearcells, which can be obtained, for example, by bone marrow aspiration;umbilical cord tissue; adipose tissue; and/or a tooth or teeth.

Therapeutic Application

The modified, DNAse-expressing cells of the present invention, includinghomospecific cells, may be administered either alone, or as acomposition (e.g., a pharmaceutical composition) in combination withdiluents and/or with other components such as cytokines,immunomodulators, other cell populations, or other small molecules orbiologics. Briefly, pharmaceutical compositions of the present inventionmay comprise a target cell population as described herein, incombination with one or more pharmaceutically or physiologicallyacceptable carriers, diluents or excipients. Such compositions maycomprise buffers such as neutral buffered saline, phosphate bufferedsaline and the like; carbohydrates such as glucose, mannose, sucrose ordextrans, mannitol; proteins such as albumin; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

Compositions of the present invention are preferably formulated forintravenous administration.

In some embodiments, the homospecific cells may be modified in vivo byadministering nucleic acid directly to the patient. In some embodiments,the nucleic acid may be administered by, for example, intraveneous,subcutaneous, intradermal, or intramuscular injection. In someembodiments, the nucleic acid may be combined with a suitable carrier.In some embodiments, the carrier may be a nanoparticle carrier, forexample a polymeric nanoparticle, lipid-based nanoparticle, or metalnanoparticle such as gold. In some embodiments, the carrier may be aviral vector, for example a lentiviral or adenoviral vector.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

The administration of the inventive compositions may be carried out inany convenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patientsubcutaneously, intradermally, intraarticularly, intratumorally,intranodally, intramedullary, intramuscularly, by intravenous injection,or intraperitoneally. In some embodiments, the inventive cellcompositions of the present invention are administered to a patient byintradermal or subcutaneous injection. In another embodiment, theinventive cell compositions of the present invention are preferablyadministered by intravenous injection.

The inventive cells and/or compositions derived thereof can beadministered to a subject for therapeutic use. Thus, the inventionprovides a method for treating or preventing a disease in a subject inneed thereof, the method comprising administering to the subject aneffective number of the inventive cells. In some embodiments, theeffective number (or amount) is determined as the number of cells. Insome embodiments, the effective number (or amount) is determined by theamount of DNAse secreted by those cells. In some embodiments, theeffective number is more than 1×10{circumflex over ( )}8 cells. In someembodiments, the effective number is more than 5×10{circumflex over( )}8 cells. In some embodiments, the effective number is more than1×10{circumflex over ( )}9 cells. In some embodiments, the effectivenumber is more than 2×10{circumflex over ( )}9 cells. In someembodiments, the effective number is more than 5×10{circumflex over( )}9 cells. In some embodiments, the effective number is more than10×10{circumflex over ( )}9 cells.

Likewise, the invention provides for use of the inventive cells for thetreatment or prevention of a disease.

In the above methods of treatment, or in the above uses, disease in needof treatment or prevention can include, for example: acute respiratorydistress syndrome (ARDS), acute kidney injury (AKI), sepsis, myocardialinfarction, systemic lupus erythematosus (SLE), rheumatoid arthritis(RA), systemic sclerosis (SS), asthma, and cancer. Some specific formsof ARDS suitable for the above methods of treatment and uses includeARDS caused, for example, by a bacterial infection such as byStreptococcus pneumoniae or Haemophilus influenzae, viral infectionssuch as influenza virus A-D or SARS-CoV-2, aspiration due to emesis orwater (e.g., near-drowning episodes), and inhalation of harmfulsubstances such as smoke or chemical fumes.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present disclosure toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

EXEMPLARY EMBODIMENTS

Some embodiments of the invention are:

-   Embodiment 1. A mammalian cell modified to secrete a DNAse enzyme.-   Embodiment 2. The cell of Embodiment 1, wherein the mammalian cell    is a human cell.-   Embodiment 3. The cell of any one of Embodiments 1-2, wherein the    mammalian cell is a white blood cell.-   Embodiment 4. The cell of Embodiment 3, wherein the white blood cell    is an NK cell.-   Embodiment 5. The cell of Embodiment 3, wherein the white blood cell    is a T cell.-   Embodiment 6. The cell of Embodiment 5, wherein the white blood cell    is a CD8+ cell.-   Embodiment 7. The cell of Embodiment 5, wherein the white blood cell    is a CD4+ cell.-   Embodiment 8. The cell of Embodiment 3, wherein the white blood cell    is a monocyte.-   Embodiment 9. The cell of Embodiment 3, wherein the white blood cell    is a macrophage.-   Embodiment 10. The cell of Embodiment 3, wherein the white blood    cell is a CD34+ cell.-   Embodiment 11. The cell of any one of Embodiments 1-2, wherein the    mammalian cell is a mesenchymal stem cell.-   Embodiment 12. The cell of any one of Embodiments 1-2, wherein the    mammalian cell is a stem cell.-   Embodiment 13. The cell of any one of Embodiments 1-12, wherein the    DNAse is DNASE1.-   Embodiment 14. The cell of any one of Embodiments 1-12, wherein the    DNAse is DNASE1L1.-   Embodiment 15. The cell of any one of Embodiments 1-12, wherein the    DNAse is DNASE1L2.-   Embodiment 16. The cell of any one of Embodiments 1-12, wherein the    DNAse is DNASE1L3.-   Embodiment 17. The cell of any one of Embodiments 1-12, wherein the    DNAse is DNASE2A.-   Embodiment 18. The cell of any one of Embodiments 1-12, wherein the    DNAse is DNASE2B.-   Embodiment 19. The cell of any one of Embodiments 1-12, wherein the    DNAse is L-DNASEII.-   Embodiment 20. The cell of any one of Embodiments 1-12, wherein the    DNAse is CAD.-   Embodiment 21. The cell of any one of Embodiments 1-13, wherein the    cell secretes two or more DNAse enzymes selected from the group    consisting of: DNASE1, DNASE1L1, DNASE1L2, DNASE1L3, DNASE2A,    DNAS2B, L-DNASEII, CAD, and EndoG.-   Embodiment 22. The cell of any one of Embodiments 1-21, wherein the    cell is modified by electroporation.-   Embodiment 23. The cell of any one of Embodiments 1-21, wherein the    cell is modified by cell squeezing.-   Embodiment 24. The cell of any one of Embodiments 1-21, wherein the    cell is modified by use of a viral vector.-   Embodiment 25. The cell of any one of Embodiments 1-24, wherein the    cell is modified by introduction of an RNA encoding the DNASE    enzyme(s).-   Embodiment 26. The cell of any one of Embodiments 1-25, wherein the    RNA is artificially enriched in pseudouridine.-   Embodiment 27. The cell of any one of Embodiments 1-25, wherein    substantially all of the uridine nucleotides in the RNA are    substituted with pseudouridine.-   Embodiment 28. The cell of any one of Embodiments 1-27, wherein the    cell is modified by introduction of an mRNA encoding the DNASE    enzyme(s).-   Embodiment 29. The cell of any one of Embodiments 1-27, wherein the    cell is modified by introduction of a self-amplifying RNA encoding    the DNASE enzyme(s).-   Embodiment 30. The cell of any one of Embodiments 1-21, wherein the    cell is modified by introduction of a nucleic acid encoding a    protein of the sequence of SEQ ID NO: 1.-   Embodiment 31. The cell of any one of Embodiments 1-21, wherein the    cell is modified by introduction of a nucleic acid encoding a    protein of the sequence of SEQ ID NO: 2.-   Embodiment 32. A cell of any one of Embodiments 1-21, wherein the    cell is modified by introduction of a nucleic acid encoding a    protein of the sequence of SEQ ID NO: 3.-   Embodiment 33. The cell of any one of Embodiments 1-24, wherein the    cell is modified by introduction of a DNA encoding the DNASE    enzyme(s).-   Embodiment 34. The cell of any one of Embodiments 1-33, wherein the    cell is modified by means of a nanoparticle, wherein the    nanoparticle comprises a nucleic acid that encodes the DNASE    enzyme(s).-   Embodiment 35. The cell of any one of Embodiments 1-34, wherein the    cell is further modified to express an anti-BCMA protein.-   Embodiment 36. The cell of Embodiment 35, wherein anti-BCMA protein    comprises a monoclonal antibody that binds BCMA.-   Embodiment 37. The cell of Embodiment 35, wherein anti-BCMA protein    comprises a bispecific antibody that binds BCMA and CD3.-   Embodiment 38. The cell of Embodiment 35, wherein anti-BCMA protein    comprises a bispecific antibody that binds BCMA and CD8.-   Embodiment 39. The cell of Embodiment 35, wherein anti-BCMA protein    comprises a bispecific antibody that binds BCMA and CD4.-   Embodiment 40. A cell therapy product comprising a plurality of    cells of any one of Embodiments 1-39.-   Embodiment 41. A cell therapy product comprising: at least one cell    of Embodiment 13 and at least one cell of Embodiment 16.-   Embodiment 42. A cell therapy product comprising at least one cell    selected of Embodiment 16 and at least one cell selected from the    group consisting of Embodiments 13, 14, 15, 17, 18, 19 and 20.-   Embodiment 43. The cell therapy product of any one of Embodiments    40-42, wherein the number of cells per dose is at least    1×10{circumflex over ( )}8.-   Embodiment 44. The cell therapy product of Embodiment 43, wherein    the number of cells per dose is at least 5×10{circumflex over ( )}8.-   Embodiment 45. The cell therapy product of Embodiment 43, wherein    the number of cells per dose is at least 1×10{circumflex over ( )}9.-   Embodiment 46. The cell therapy product of Embodiment 43, wherein    the number of cells per dose is at least 5×10{circumflex over ( )}9.-   Embodiment 47. The cell therapy product of Embodiment 43, wherein    the number of cells per dose is at least 1×10{circumflex over    ( )}10.-   Embodiment 48. A method of treating a subject in need thereof,    comprising administering to the subject a cell of any one of    Embodiments 1-39.-   Embodiment 49. A method of treating a subject in need thereof,    comprising administering to the subject a cell therapy product of    any one of Embodiments 40-47.-   Embodiment 50. The method of any one of Embodiments 48-49, wherein    the subject suffers from Acute Respiratory Distress Syndrome.-   Embodiment 51. The method of any one of Embodiments 48-49, wherein    the subject suffers from a viral infection.-   Embodiment 52. The method of any one of Embodiments 48-49, wherein    the subject suffers from COVID-19.-   Embodiment 53. The method of any one of Embodiments 48-49, wherein    the subject suffers from acute kidney injury.-   Embodiment 54. The method of any one of Embodiments 48-49, wherein    the subject suffers from sepsis.-   Embodiment 55. The method of any one of Embodiments 48-49, wherein    the subject suffers from myocardial infarction.-   Embodiment 56. The method of any one of Embodiments 48-49, wherein    the subject suffers from acute ischemia.-   Embodiment 57. The method of any one of Embodiments 48-49, wherein    the subject suffers from systemic lupus erythematosus.-   Embodiment 58. The method of any one of Embodiments 48-49, wherein    the subject suffers from rheumatoid arthritis.-   Embodiment 59. The method of any one of Embodiments 48-49, wherein    the subject suffers from inflammatory bowel disease.-   Embodiment 60. The method of any one of Embodiments 48-49, wherein    the subject suffers from cancer.-   Embodiment 61. Use of a cell of any one of Embodiments 1-39 for    treatment of disease.-   Embodiment 62. Use of a cell therapy product of any one of    Embodiments 40-47 for treatment of disease.-   Embodiment 63. The use of any one of Embodiments 61-62, wherein the    disease is Acute

Respiratory Distress Syndrome.

-   Embodiment 64. The use of any one of Embodiments 61-62, wherein the    disease is a viral infection.-   Embodiment 65. The use of any one of Embodiments 61-62, wherein the    disease is COVID-19.-   Embodiment 66. The use of any one of Embodiments 61-62, wherein the    disease is acute kidney injury.-   Embodiment 67. The use of any one of Embodiments 61-62, wherein the    disease is sepsis.-   Embodiment 68. The use of any one of Embodiments 61-62, wherein the    disease is myocardial infarction.-   Embodiment 69. The use of any one of Embodiments 61-62, wherein the    disease is acute ischemia.-   Embodiment 70. The use of any one of Embodiments 61-62, wherein the    disease is systemic lupus erythematosus.-   Embodiment 71. The use of any one of Embodiments 61-62, wherein the    disease is rheumatoid arthritis.-   Embodiment 72. The use of any one of Embodiments 61-62, wherein the    disease is inflammatory bowel disease.-   Embodiment 73. The use of any one of Embodiments 61-62, wherein the    disease is cancer.-   Embodiment 74. A nanoparticle comprising a nucleic acid encoding a    DNAse, wherein the nanoparticle is adapted for combination with a    mammalian cell, and whereby the combination results in secretion by    the mammalian cell of the DNAse.-   Embodiment 75. The nanoparticle of Embodiment 74, wherein the DNAse    is DNASE1.-   Embodiment 76. The nanoparticle of Embodiment 74, wherein the DNAse    is DNASE1L3.-   Embodiment 77. The nanoparticle of Embodiment 74, wherein the    combination results in secretion by the mammalian cell of DNASE1 and    DNASE1L3.-   Embodiment 78. The nanoparticle of any one of Embodiments 74-77,    wherein the nucleic acid is DNA.-   Embodiment 79. The nanoparticle of any one of Embodiments 74-77,    wherein the nucleic acid is RNA.-   Embodiment 80. The nanoparticle of Embodiment 79, wherein the RNA is    mRNA.-   Embodiment 81. The nanoparticle of Embodiment 79, wherein the RNA is    saRNA.-   Embodiment 82. The nanoparticle of any one of Embodiments 74-81,    wherein the combination occurs in vitro.-   Embodiment 83. The nanoparticle of any one of Embodiments 74-81,    wherein the combination occurs in vivo.-   Embodiment 84. The nanoparticle of Embodiment 74, further comprising    a nucleic acid comprising an anti-BCMA protein, whereby the    combination further results in secretion by the cell of the    anti-BCMA protein.-   Embodiment 85. A polynucleotide comprising the structure of    Construct A.-   Embodiment 86. A polynucleotide comprising the structure of    Construct B.-   Embodiment 87. A polynucleotide comprising the structure of    Construct C.-   Embodiment 88. A polynucleotide comprising the structure of    Construct D.-   Embodiment 89. A polynucleotide comprising the structure of    Construct E.-   Embodiment 90. A polynucleotide comprising the structure of    Construct F.-   Embodiment 91. A polynucleotide comprising the structure of    Construct G.-   Embodiment 92. A polynucleotide comprising the structure of    Construct H.-   Embodiment 93. A polynucleotide comprising the structure of    Construct I.-   Embodiment 94. The polynucleotide of any one of Embodiments 85-93,    wherein the polynucleotide is artificially enriched in    pseudouridine.-   Embodiment 95. The polynucleotide of any one of Embodiments 85-93,    wherein substantially all of the uridine nucleotides in the RNA are    substituted with pseudouridine.-   Embodiment 96. A mammalian cell comprising a polynucleotide    comprising a structure selected from the group consisting of:    Construct A, Construct B, Construct C, Construct D, Construct E,    Construct F, Construct G, Construct H, and Construct I.-   Embodiment 97. The cell of Embodiment 96, wherein the polynucleotide    is an RNA that is artificially enriched in pseudouridine.-   Embodiment 98. The polynucleotide of Embodiment 96, wherein the    polynucleotide is an RNA wherein substantially all of the uridine    nucleotides in the RNA are substituted with pseudouridine.-   Embodiment 99. A mammalian cell comprising a polynucleotide having    the structure of Construct A.-   Embodiment 100. A mammalian cell comprising a polynucleotide having    the structure of Construct B.-   Embodiment 101. A mammalian cell comprising a polynucleotide having    the structure of Construct C.-   Embodiment 102. A mammalian cell comprising a polynucleotide having    the structure of Construct D.-   Embodiment 103. A mammalian cell comprising a polynucleotide having    the structure of Construct E.-   Embodiment 104. A mammalian cell comprising a polynucleotide having    the structure of Construct F.-   Embodiment 105. A mammalian cell comprising a polynucleotide having    the structure of Construct G.-   Embodiment 106. A mammalian cell comprising a polynucleotide having    the structure of Construct H.-   Embodiment 107. A mammalian cell comprising a polynucleotide having    the structure of Construct I.-   Embodiment 108. The cell of any one of Embodiments 99-107, wherein    the cell is an NK cell.-   Embodiment 109. The cell of any one of Embodiments 99-107, wherein    the cell is an MSC.-   Embodiment 110. The cell of any one of Embodiments 99-107, wherein    the cell is a T cell.-   Embodiment 111. The cell of any one of Embodiments 99-107, wherein    the cell is a monocyte.-   Embodiment 112. The cell of any one of Embodiments 99-107, wherein    the cell is a macrophage.-   Embodiment 113. The cell of any one of Embodiments 99-107, wherein    the cell is a stem cell.-   Embodiment 114. The cell of any one of Embodiments 99-113, wherein    the polynucleotide is an RNA that is artificially enriched in    pseudouridine.-   Embodiment 115. The cell of any one of Embodiments 99-113, wherein    the polynucleotide is an RNA wherein substantially all of the    uridine nucleotides in the RNA are substituted with pseudouridine.-   Embodiment 116. A mammalian cell comprising a first exogenous RNA    encoding DNASE1 and a second exogenous RNA encoding DNASE1L3,    wherein the second exogenous RNA is artificially enriched in    pseudouridine.-   Embodiment 117. A mammalian cell comprising a first exogenously    introduced RNA encoding DNASE1 and a second exogenously introduced    RNA encoding DNASE1L3, wherein substantially all of the uridine    nucleotides in the second exogenously introduced RNA are substituted    with pseudouridine.-   Embodiment 118. The cell of any one of Embodiments 116-117, wherein    the RNA encoding DNASE1 is not artificially enriched in    pseudouridine.-   Embodiment 119. A mammalian mesenchymal stem cell comprising an    exogenous RNA encoding DNASE1.-   Embodiment 120. A mammalian mesenchymal stem cell comprising an    exogenous RNA encoding DNASE1L3.-   Embodiment 121. A mammalian mesenchymal stem cell comprising an    exogenous RNA encoding DNASE1 and DNASE1L3.-   Embodiment 122. A mammalian mesenchymal stem cell comprising a first    exogenous RNA encoding DNASE1 and a second exogenous DNASE1L3.-   Embodiment 123. The cell of Embodiment 122, wherein the second    exogenous RNA is artificially enriched in pseudouridine.-   Embodiment 124. The cell of Embodiment 122, wherein substantially    all of the uridine nucleotides in the second exogenously introduced    RNA are substituted with pseudouridine.-   Embodiment 125. The cell of any one of Embodiments 122-124, wherein    the first RNA is not artificially enriched in pseudouridine.-   Embodiment 126. The cell of any one of Embodiments 99-125, wherein    the cell is a human cell.-   Embodiment 127. A method for treating or preventing disease, the    method comprising administering to a subject in need thereof a cell    of any one of Embodiments 99-125.-   Embodiment 128. Use of a cell of any one of Embodiments 99-125 for    the treatment or prevention of a disease.-   Embodiment 129. The method of any one of Embodiments 127-128,    wherein the disease is Acute Respiratory Distress Syndrome.-   Embodiment 130. The method of any one of Embodiments 127-128,    wherein the disease is a viral infection.-   Embodiment 131. The method of any one of Embodiments 127-128,    wherein the disease is COVID-19.-   Embodiment 132. The method of any one of Embodiments 127-128,    wherein the disease is acute kidney injury.-   Embodiment 133. The method of any one of Embodiments 127-128,    wherein the disease is sepsis.-   Embodiment 134. The method of any one of Embodiments 127-128,    wherein the disease is myocardial infarction.-   Embodiment 135. The method of any one of Embodiments 127-128,    wherein the disease is acute ischemia.-   Embodiment 136. The method of any one of Embodiments 127-128,    wherein the disease is systemic lupus erythematosus.-   Embodiment 137. The method of any one of Embodiments 127-128,    wherein the disease is rheumatoid arthritis.-   Embodiment 138. The method of any one of Embodiments 127-128,    wherein the disease is inflammatory bowel disease.-   Embodiment 139. The method of any one of Embodiments 127-128,    wherein the disease is cancer.

EXAMPLES

Without further elaboration, it is believed that one of ordinary skillin the art can, based on the above description, utilize the presentdisclosure to its fullest extent. Nevertheless, in order that theinvention described herein may be more fully understood, the followingexamples are set forth. The synthetic examples described in thisapplication are offered to illustrate the compounds and methods providedherein and are not to be construed in any way as limiting their scope.All publications cited herein are incorporated by reference for thepurposes or subject referenced herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein are meant to be non-limiting.In describing and claiming the present invention, the followingterminology will be used.

Example 1: Production of Functional DNAse-Secreting NK Cells from anInventive saRNA Construct

The following example is of human DNAse-secreting NK cells produced byintroduction of an inventive saRNA construct that encodes human DNASE1and DNASE1L3.

An inventive saRNA construct comprising the nucleotide sequence of SEQID NO: 3 is generated by in vitro transcription from a DNA plasmid. Thein vitro transcription is performed by SP6 RNA polymerase from alinearized plasmid template (alternatively, a T7 polymerase can beused). A polyadenine tail of about 150 adenine nucleotides can be addedenzymatically to the saRNA. A 7-methylguanosine cap can be incorporatedat the 5′ end of the saRNA during the co-transcriptional RNA synthesis.

This inventive saRNA construct comprises, from 5′ to 3′: a 5′ cap, a 5′UTR described as SEQ ID NO: 15, an RNA-dependent RNA polymerase (RDRP)sequence described as SEQ ID NO: 17 (i.e., NSP1-NSP4), a DNASE1 openreading frame (ORF) described as SEQ ID NO: 1, a T2A sequence describedin SEQ ID NO: 14, a DNASE1L3 ORF described as SEQ ID NO: 2, an T2Asequence described in SEQ ID NO: 14, a translation enhancer ORFdescribed as SEQ ID NO: 8, a T2A sequence described in SEQ ID NO: 14, ahuman IL-15 ORF described as SEQ ID NO: 4, a 3′ UTR described as SEQ IDNO: 16, and a 3′ polyadenine tail of 150 adenine units or more.

A translation enhancer mRNA construct encoding a protein comprising thesequence of SEQ ID NO: 8 is generated by in vitro transcription from aDNA plasmid. The in vitro transcription can be performed by T7 RNApolymerase from a linearized plasmid template. A polyadenine tail ofabout 150 adenine nucleotides is added enzymatically to the mRNA. A7-methylguanosine cap is incorporated at the 5′ end of the mRNA duringthe co-transcriptional RNA synthesis. The translation enhancer mRNAconstruct comprises, from 5′ to 3′: a 5′ cap, a 5′ UTR described as SEQID NO: 23, a Kozak sequence described in SEQ ID NO: 20, an open readingframe (ORF) of sequence of SEQ ID NO: 8, a 3′ UTR described as SEQ IDNO: 24, and a 3′ polyadenine tail of 150 adenine units or more.

To prepare NK cells with the RNA constructs, NK cells are isolated fromumbilical cord blood by CD3+ magnetic bead negative selection followedby CD56+ magnetic bead positive selection to obtain >98% pure NK cellsthat are >95% viable. These NK cells are expanded by incubation at 37°C. with 5% CO₂ in the presence of K562 feeder cells engineered toexpress membrane-bound IL-15 for about 14 days. The cells areresuspended in transfection buffer and transfected with a mixture of thesaRNA construct and mRNA construct by electroporation (4D NUCLEOFECTOR®,Lonza) according to manufacturer's instructions. The cells are thenreturned to culture in a standard medium containing IL-15 for overnightincubation, then frozen at −80 C. Transfected cells are thawed andincubated for another 7 days in the presence of complete mediumcontaining IL-15 and supernatant samples are collected and frozen at 4h, 1 day, 2 days, 3 days, 5 days and 7 days after thaw. Thawed cells andsupernatants are assayed for viability and activity according to themethods described below.

NK cells obtained from the above-described process can be tested forviability, DNASE1 and DNASE1L3 expression, and DNA- andchromatin-degrading capacity. Viability can be determined by flowcytometry on a GUAVA® EASYCYTE® 12HT Flow cytometer (EMD Millipore). Totest viability, a sample of the NK cells is mixed with propidium iodideand run on the flow cytometer with electronic gating on fluorescence inthe near infrared channel. To test expression of DNASE1 and DNASE1L3,commercially available DNASE1 and DNASE1L3 kits (i.e., Abbexa) are usedto assay supernatants of cultured NK cells according to themanufacturers' instructions. To test DNA degrading capacity oftransfected NK cells, culture supernatants are tested with afluorometric DNASE1 assay kit (i.e., AbCam) according to themanufacturer's instructions. To test chromatin degrading capacity oftransfected NK cells, culture supernatants are first incubated with NETsgenerated from phorbol myristate acetate-activated human neutrophils.Neutrophils are isolated from fresh apheresis product by density gradecentrifugation. Recombinant human DNASE1 and DNASE1L3 are used aspositive controls. The amount of NET DNA released is determined byadding picogreen (Invitrogen), a DNA fluorescence dye, to the mixedculture and then quantified by fluorescence spectrometry.

It is expected that DNASE1- and DNASE1L3-transfected NK cells willexpress and secrete functional DNASE1 and DNASE1L3 constitutively overthe course of at least 24 hours. It is further expected that NK cellswill maintain the capacity to express and secrete functional DNASE1 andDNASE1L3 following freeze/thaw.

Thus, NK cells can be transfected with the inventive saRNA sequence toexpress and secrete functional DNASE1 and DNASE1L3 protein. The resultof this process is inventive, i.e., DNase-secreting NK cells that areuseful for therapeutic administration, e.g., to a person affected byARDS.

Example 2: Production of Functional DNAse-Secreting MSCs from an saRNAConstruct

The following example is of human DNAse-secreting MSCs produced byintroduction of an inventive saRNA construct that encodes human DNASE1and DNASE1L3.

An inventive saRNA construct comprising the nucleotide sequence of SEQID NO: 3 is generated by in vitro transcription from a DNA plasmid. Thein vitro transcription is performed by SP6 RNA polymerase from alinearized plasmid template (alternatively, a T7 polymerase can beused). A polyadenine tail of about 150 adenine nucleotides is addedenzymatically to the saRNA. A 7-methylguanosine cap is incorporated atthe 5′ end of the saRNA during the co-transcriptional RNA synthesis.

The inventive saRNA construct comprise, from 5′ to 3′: a 5′ cap, a 5′UTR described as SEQ ID NO: 15, an RNA-dependent RNA polymerase (RDRP)sequence described as SEQ ID NO: 17, DNASE1 open reading frame (ORF)described as SEQ ID NO: 1, a T2A sequence described in SEQ ID NO: 14, aDNASE1L3 ORF described as SEQ ID NO: 2, a T2A sequence described in SEQID NO: 14, a translation enhancer ORF described as SEQ ID NO: 8, a 3′UTR described as SEQ ID NO: 16, and a 3′ polyadenine tail of 150 adenineunits or more.

A translation enhancer mRNA construct encoding a protein comprising thesequence of SEQ ID NO: 8 is generated by in vitro transcription from aDNA plasmid. The in vitro transcription is performed by T7 RNApolymerase from a linearized plasmid template. A polyadenine tail ofabout 150 adenine nucleotides is added enzymatically to the mRNA. A7-methylguanosine cap is incorporated at the 5′ end of the mRNA duringthe co-transcriptional RNA synthesis. The translation enhancer mRNAconstruct comprises, from 5′ to 3′: a 5′ cap, a 5′ UTR described as SEQID NO: 15, a Kozak sequence described in SEQ ID NO: 20, the translationenhancer open reading frame (ORF) comprising the nucleotide sequence ofSEQ ID NO: 8, a 3′ UTR described as SEQ ID NO: 16, and a 3′ polyadeninetail of 150 adenine units or more.

To prepare MSCs from RNA constructs, MSCs isolated from human umbilicalcord are purchased from ATCC, thawed and expanded in T75 flasks byculture at 37° C. with 5% CO₂ in α-DMEM medium supplemented with 10%human serum. Cells are passaged upon reaching 80% confluency. The cellsare resuspended in transfection buffer and transfected with a mixture ofthe saRNA construct and mRNA construct by electroporation (4DNUCLEOFECTOR®, Lonza) according to manufacturer's instructions. Thecells are then returned to culture in complete medium for overnightincubation, then frozen at −80 C. Transfected cells are thawed andincubated for another 7 days in the presence of complete medium andsupernatant samples are collected and frozen at 4 h, 1 day, 2 days, 3days, 5 days and 7 days after thaw. Thawed cells and supernatants areassayed for viability and activity according to the methods describedbelow.

MSCs obtained from the above-described process can be tested forviability, DNASE1 and DNASE1L3 expression, and DNA- andchromatin-degrading capacity. For example, viability is determined byflow cytometry on a GUAVA® EASYCYTE® 12HT Flow cytometer (EMDMillipore). To test viability, a sample of the MSCs is mixed withpropidium iodide and run on the flow cytometer with electronic gating onfluorescence in the near infrared channel. To test expression of DNASE1and DNASE1L3, commercially available DNASE1 and DNASE1L3 kits (i.e.,Abbexa) are used to assay supernatants of cultured MSCs according to themanufacturers' instructions. To test DNA degrading capacity oftransfected MSCs, culture supernatants are tested with a fluorometricDNASE1 assay kit (i.e., AbCam) according to the manufacturer'sinstructions. To test chromatin degrading capacity of transfected MSCs,culture supernatants are first incubated with NETs generated fromphorbol myristate acetate-activated human neutrophils. Neutrophils areisolated from fresh apheresis product by density grade centrifugation.Recombinant human DNASE1 and DNASE1L3 are used as positive controls. Theamount of NET DNA released is determined by adding picogreen(Invitrogen), a DNA fluorescence dye, to the mixed culture and thenquantified by fluorescence spectrometry.

It is expected that DNASE1- and DNASE1L3-transfected MSCs will expressand secrete functional DNASE1 and DNASE1L3 constitutively over thecourse of at least 24 hours. It is further expected that MSCs willmaintain the capacity to express and secrete functional DNASE1 andDNASE1L3 following freeze/thaw.

Thus, MSCs can be transfected with the inventive saRNA sequence toexpress and secrete functional DNASE1 and DNASE1L3 protein. The resultof this process is inventive, i.e., DNase-secreting MSCs that are usefulfor therapeutic administration, e.g., to a person affected by ARDS.

Example 3: Production of Functional DNAse-Secreting NK Cells from anInventive Combination of mRNA Constructs

The following example is of human DNAse-secreting NK cells produced byintroduction of an inventive combination of mRNA constructs that encodehuman DNASE1 and DNASE1L3.

Four separate mRNA constructs, corresponding respectively to SEQ ID NO:1 (DNASE1), SEQ ID NO: 2 (DNASE1L3), SEQ ID NO: 4 (transmembrane IL-15)and SEQ ID NO: 8 (translation enhancer E3), are generated by in vitrotranscription from DNA plasmids. The in vitro transcription is performedby T7 RNA polymerase from a linearized plasmid template. A polyadeninetail of about 150 adenine nucleotides is added enzymatically to eachmRNA. A 7-methylguanosine cap is incorporated at the 5′ end of each mRNAduring the co-transcriptional RNA synthesis.

The first mRNA construct comprises, from 5′ to 3′: a 5′ cap; a 5′ UTRdescribed as SEQ ID NO: 23; a Kozak sequence described in SEQ ID NO: 20;a sequence of SEQ ID NO:1; a 3′ UTR described as SEQ ID NO: 24, and a 3′polyadenine tail of 150 adenine units or more.

The second mRNA construct comprises, from 5′ to 3′: a 5′ cap; a 5′ UTRdescribed as SEQ ID NO: 23; a Kozak sequence described in SEQ ID NO: 20;a sequence of SEQ ID NO:2; a 3′ UTR described as SEQ ID NO: 24, and a 3′polyadenine tail of 150 adenine units or more.

The third mRNA construct comprises, from 5′ to 3′: a 5′ cap; a 5′ UTRdescribed as SEQ ID NO: 23; a Kozak sequence described in SEQ ID NO: 20;a sequence of SEQ ID NO:4; a 3′ UTR described as SEQ ID NO: 24, and a 3′polyadenine tail of 150 adenine units or more.

The fourth mRNA construct comprises, from 5′ to 3′: a 5′ cap; a 5′ UTRdescribed as SEQ ID NO: 23; a Kozak sequence described in SEQ ID NO: 20;a sequence of SEQ ID NO:8; a 3′ UTR described as SEQ ID NO: 24, and a 3′polyadenine tail of 150 adenine units or more.

To prepare NK cells from mRNA constructs, NK cells are isolated fromumbilical cord blood by CD3+ magnetic bead negative selection followedby CD56+ magnetic bead positive selection to obtain >98% pure NK cellsthat are >95% viable. These NK cells are expanded by incubation at 37°C. with 5% CO₂ in the presence of K562 feeder cells engineered toexpress membrane-bound IL-15 for about 14 days. The cells areresuspended in transfection buffer and simultaneously transfected with amixture of the four aforementioned mRNA constructs by electroporation(4D NUCLEOFECTOR®, Lonza) according to manufacturer's instructions. Thecells are then returned to culture in a standard medium containing IL-15for overnight incubation, and then are frozen at −80 C. Transfectedcells are thawed and incubated for another 7 days in the presence ofcomplete medium containing IL-15 and supernatant samples are collectedand frozen at 4 h, 1 day, 2 days, 3 days, 5 days and 7 days after thaw.Thawed cells and supernatants are assayed for viability and activityaccording to the methods described below.

NK cells obtained from the above-described process can be tested forviability, DNASE1 and DNASE1L3 expression, and DNA- andchromatin-degrading capacity. As an example, viability is determined byflow cytometry on a GUAVA® EASYCYTE® 12HT Flow cytometer (EMDMillipore). To test viability, a sample of the NK cells is mixed withpropidium iodide and run on the flow cytometer with electronic gating onfluorescence in the near infrared channel. To test expression of DNASE1and DNASE1L3, commercially available DNASE1 and DNASE1L3 kits (i.e.,Abbexa) are used to assay supernatants of cultured NK cells according tothe manufacturers' instructions. To test DNA degrading capacity oftransfected NK cells, culture supernatants are tested with afluorometric DNASE1 assay kit (i.e., AbCam) according to themanufacturer's instructions. To test chromatin degrading capacity oftransfected NK cells, culture supernatants are first incubated with NETsgenerated from phorbol myristate acetate-activated human neutrophils.Neutrophils are isolated from fresh apheresis product by density gradecentrifugation. Recombinant human DNASE1 and DNASE1L3 are used aspositive controls. The amount of NET DNA released is determined byadding picogreen (Invitrogen), a DNA fluorescence dye, to the mixedculture and then quantified by fluorescence spectrometry.

It is expected that the NK cells transfected with the above-describedconstructs will express and secrete functional DNASE1 and DNASE1L3constitutively over the course of at least 24 hours. It is furtherexpected that NK cells will maintain the capacity to express and secretefunctional DNASE1 and DNASE1L3 following freeze/thaw.

Thus, NK cells can be transfected with the inventive combination of mRNAsequences to express and secrete functional DNASE1 and DNASE1L3 protein.The result of this process is inventive, DNase-secreting, NK cells thatare useful for therapeutic administration, e.g., to a person affected byARDS.

Example 4: Production of Functional DNAse-Secreting MSCs from anInventive Combination of mRNA Constructs

The following example is of human DNAse-secreting MSCs produced byintroduction of an inventive combination of mRNA construct that encodehuman DNASE1 and DNASE1L3.

Three separate mRNA constructs, corresponding respectively to SEQ ID NO:1 (DNASE1), SEQ ID NO: 2 (DNASE1L3), and SEQ ID NO: 8 (translationenhancer E3), are generated by in vitro transcription from DNA plasmids.The in vitro transcription is performed by T7 RNA polymerase from alinearized plasmid template. A polyadenine tail of about 150 adeninenucleotides is added enzymatically to each mRNA. A 7-methylguanosine capis incorporated at the 5′ end of each mRNA during the co-transcriptionalRNA synthesis.

The first mRNA construct comprises, from 5′ to 3′: a 5′ cap; a 5′ UTRdescribed as SEQ ID NO: 23; a Kozak sequence described in SEQ ID NO: 20;a sequence of SEQ ID NO:1; a 3′ UTR described as SEQ ID NO: 24, and a 3′polyadenine tail of 150 adenine units or more.

The second mRNA construct comprises, from 5′ to 3′: a 5′ cap; a 5′ UTRdescribed as SEQ ID NO: 23; a Kozak sequence described in SEQ ID NO: 20;a sequence of SEQ ID NO:2; a 3′ UTR described as SEQ ID NO: 24, and a 3′polyadenine tail of 150 adenine units or more.

The third mRNA construct comprises, from 5′ to 3′: a 5′ cap; a 5′ UTRdescribed as SEQ ID NO: 23; a Kozak sequence described in SEQ ID NO: 20;a sequence of SEQ ID NO:8; a 3′ UTR described as SEQ ID NO: 24, and a 3′polyadenine tail of 150 adenine units or more.

To prepare MSCs from RNA constructs, MSCs isolated from human umbilicalcord are purchased from ATCC, thawed and expanded in T75 flasks byculture at 37° C. with 5% CO₂ in α-DMEM medium supplemented with 10%human serum. The cells are passaged upon reaching 80% confluency. Thecells are resuspended in transfection buffer and simultaneouslytransfected with a mixture of the three aforementioned mRNA constructsby electroporation (4D NUCLEOFECTOR®, Lonza) according to manufacturer'sinstructions. The cells are then returned to culture in complete mediumfor overnight incubation, then frozen at −80 C. Transfected cells arethawed and incubated for another 7 days in the presence of completemedium and supernatant samples are collected and frozen at 4 h, 1 day, 2days, 3 days, 5 days and 7 days after thaw. Thawed cells andsupernatants are assayed for viability and activity according to themethods described below.

MSCs obtained from the above-described process can be tested forviability, DNASE1 and DNASE1L3 expression, and DNA- andchromatin-degrading capacity. As an example, viability is determined byflow cytometry on a GUAVA® EASYCYTE® 12HT Flow cytometer (EMDMillipore). To test viability, a sample of the MSCs is mixed withpropidium iodide and run on the flow cytometer with electronic gating onfluorescence in the near infrared channel. To test expression of DNASE1and DNASE1L3, commercially available DNASE1 and DNASE1L3 kits (i.e.,Abbexa) are used to assay supernatants of cultured MSCs according to themanufacturers' instructions. To test DNA degrading capacity oftransfected MSCs, culture supernatants are tested with a fluorometricDNASE1 assay kit (i.e., AbCam) according to the manufacturer'sinstructions. To test chromatin degrading capacity of transfected MSCs,culture supernatants are first incubated with NETs generated fromphorbol myristate acetate-activated human neutrophils. Neutrophils areisolated from fresh apheresis product by density grade centrifugation.Recombinant human DNASE1 and DNASE1L3 are used as positive controls. Theamount of NET DNA released is determined by adding picogreen(Invitrogen), a DNA fluorescence dye, to the mixed culture and thenquantified by fluorescence spectrometry.

It is expected that the MSCs transfected with the above-describedconstructs will express and secrete functional DNASE1 and DNASE1L3constitutively over the course of at least 24 hours. It is furtherexpected that MSCs will maintain the capacity to express and secretefunctional DNASE1 and DNASE1L3 following freeze/thaw.

Thus, MSCs can be transfected with the inventive combination of mRNAsequences to express and secrete functional DNASE1 and DNASE1L3 protein.The result of this process is inventive, i.e., DNase-secreting, MSCsthat are useful for therapeutic administration, e.g., to a personaffected by ARDS.

Example 5: In Vivo Safety and Efficacy of Functional DNAse-SecretingMSCs in a Mouse Model of ALI

DNAse-secreting MSCs produced from inventive RNA constructs comprisingthe nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, were testedin an Acute Lung Injury (ALI) animal model of ARDS. In this model, ALIis first induced by the intratracheal administration oflipopolysaccharide (LPS), and MSCs are then administered by intravenousinjection.

DNAse-secreting MSCs were prepared by transfection of the inventive RNAconstructs, as discussed in Example 2. 8-12 week old C57BL/6 mice wereanesthetized and administered 2 mg/kg of LPS solution by anintratracheal catheter. At 4 hours after LPS administration, mice wererandomized to receive intravenous vehicle only (negative control), 1×10⁶untransfected MSCs (negative control), or 1×10⁶ DNAse-secreting MSCs. 5animals were assigned to each group.

12 hours after MSC administration, mice were anesthetized andbronchoalveolar lavage fluid (BALF) was collected with 1 mL phosphatebuffered saline (PBS) solution. 0.5 mL of BALF was frozen withoutfixation for analysis of TNF-α, IL-6 and IL-10 by commercially availablemurine ELISA kits or for NET formation by Quant-iT dsDNA HS kit(Invitrogen). 0.5 mL of BALF was collected into Cyto-Chex® BCT Tubes(Streck) for analysis of total cell infiltrate and infiltration ofspecific cell types (i.e., macrophages, neutrophils and lymphocytes) byflow cytometry.

Two mice from each group were sacrificed 24 hours after LPSadministration and lung tissue was collected. Each lung was fixed in 4%formalin solution and processed for H&E staining. The remaining animalswere followed for up to 5 days before being sacrificed and processed asabove Inflammation scores were quantified with Image Pro Plus software(Media Cybernetics).

The DNAse-secreting MSC group is expected to show fewer inflammatorycells from BALF, lower amounts of TNF-α and IL-6 from BALF, less NETformation, and lower inflammatory scores on histology compared with theuntransfected MSC group and vehicle only-treated animals. Furthermore,animals in the DNAse-secreting MSC group are expected to survive longercompared with the control groups.

Thus, DNAse-secreting MSCs are expected to reduce ALI and improvesurvival in a murine model of ARDS.

Example 6: Clinical Safety and Efficacy of Functional DNAse-SecretingMSCs in Patients with ARDS

A randomized clinical trial is conducted to test inventive,DNAse-secreting MSCs in patients with ARDS. The clinical trial enrolls20 patients who meet the following criteria: at least 18 years old; ARDSper Berlin Criteria; and current endotracheal intubation with mechanicalventilation. The patients are randomized in a 1:1 ratio to receivestandard of care with or without DNAse-secreting MSCs. DNAse-secretingMSCs are made according to Example 2, supra. For those patients thusrandomized, the DNAse-secreting MSCs are administered at a dose of about1.1×10⁹ cells daily for three successive days. It is expected thatfollowing the initiation of treatment, e.g., 24 to 96 hours after thefirst dose, patients who receive the MSCs will, compared to those who donot receive the MSCs, show more rapid improvement of clinical status asmeasured by PaO2/FiO2 (ratio of arterial partial pressure of O2 tofraction of inspired O2), time to extubation, and survival.

Example 7: Production of Bifunctional Anti-BCMA Bispecific Antibody andDNAse-Secreting MSCs from an Inventive saRNA Construct

The following example is of MSCs modified to secrete a DNAse enzyme anda bispecific antibody. Namely, the MSCs of this example are human MSCsmodified by the introduction of saRNA to express human DNASE1, humanDNASE1L3, and a bispecific antibody directed to human BCMA and humanCD3. The bispecific antibody of this example is also referred to as abispecific T-cell engager.

An inventive saRNA construct, described hereunder, is generated by invitro transcription from a DNA plasmid. The in vitro transcription isperformed by SP6 RNA polymerase from a linearized plasmid template. Apolyadenine tail of about 150 adenine nucleotides is added enzymaticallyto the saRNA. A 7-methylguanosine cap is incorporated at the 5′ end ofthe saRNA during the co-transcriptional RNA synthesis.

The inventive saRNA construct comprises, from 5′ to 3′: a 5′ cap, a 5′UTR described as SEQ ID NO: 15, an RNA-dependent RNA polymerase (RDRP)sequence described as SEQ ID NO: 17, DNASE1 open reading frame (ORF)described as SEQ ID NO: 1, a T2A sequence described in SEQ ID NO: 14, aDNASE1L3 ORF described as SEQ ID NO: 2, a T2A sequence described as SEQID NO: 14, an anti-BCMA-CD3 bispecific antibody ORF described as SEQ IDNO: 21, a T2A sequence described in SEQ ID NO: 14, a translationenhancer ORF described as SEQ ID NO: 8, a 3′ UTR described as SEQ ID NO:16, and a 3′ polyadenine tail of 150 adenine units or more. Theconstruct can be represented as follows:

Cap 5′ UTR NSP1-NSP4 26S promoter C protein DNASE1L3 T2A DNASE1 T2ABispec 3′ UTR polyA

A translation enhancer mRNA construct described by sequence of SEQ IDNO: 8 is generated by in vitro transcription from a DNA plasmid. The invitro transcription is performed by SP6 RNA polymerase from a linearizedplasmid template. A polyadenine tail of about 150 adenine nucleotides isadded enzymatically to the mRNA. A 7-methylguanosine cap is incorporatedat the 5′ end of the mRNA during the co-transcriptional RNA synthesis.The translation enhancer mRNA construct comprises, from 5′ to 3′: a 5′cap, a 5′ UTR described as SEQ ID NO: 15, a Kozak sequence described inSEQ ID NO: 20, the open reading frame (ORF) described by the sequence ofSEQ ID NO: 8, a 3′ UTR described as SEQ ID NO: 16, and a 3′ polyadeninetail of 150 adenine units or more.

To prepare MSCs from RNA constructs, MSCs isolated from human umbilicalcord are purchased from ATCC, thawed and expanded in T75 flasks byculture at 37° C. with 5% CO₂ in DMEM medium supplemented with 10% humanserum. The cells are passaged upon reaching 80% confluency. The cellsare resuspended in transfection buffer and transfected with a mixture ofthe saRNA construct and mRNA construct by electroporation (4DNUCLEOFECTOR®, Lonza) according to manufacturer's instructions. Thecells are then returned to culture in complete medium for overnightincubation, then frozen at −80° C. The transfected cells are thawed andincubated for another 7 days in the presence of complete medium andsupernatant samples are collected and frozen at 4 h, 1 day, 2 days, 3days, 5 days and 7 days after thaw. Thawed cells and supernatants can beassayed for viability and activity according to the methods describedbelow.

MSCs obtained from the above-described process can be tested forviability, DNASE1 and DNASE1L3 expression, DNA- and chromatin-degradingcapacity. As an example, viability is determined by flow cytometry on aGUAVA® EASYCYTE® 12HT Flow cytometer (EMD Millipore). To test viability,a sample of the MSCs is mixed with propidium iodide and run on the flowcytometer with electronic gating on fluorescence in the near infraredchannel. To test expression of DNASE1 and DNASE1L3, commerciallyavailable DNASE1 and DNASE1L3 kits (i.e., Abbexa) are used to assaysupernatants of cultured MSCs according to the manufacturers'instructions. To test DNA degrading capacity of transfected MSCs,culture supernatants are tested with a fluorometric DNASE1 assay kit(i.e., AbCam) according to the manufacturer's instructions. To testchromatin degrading capacity of transfected MSCs, culture supernatantsare first incubated with NETs generated from phorbol myristateacetate-activated human neutrophils. Neutrophils are isolated from freshapheresis product by density grade centrifugation. Recombinant humanDNASE1 and DNASE1L3 are used as positive controls. The amount of NET DNAreleased is determined by adding picogreen (Invitrogen), a DNAfluorescence dye, to the mixed culture and then quantified byfluorescence spectrometry.

MSCs obtained from the above-described process were tested for theircapacity to secrete functional anti-BCMA-CD3 bispecific antibody, i.e.,the ability to kill BCMA+ myeloma (tumor) cells in the presence of naïveCD3+ T-cells. To test the capacity of MSCs to secrete anti-BCMA-CD3bispecific antibody, supernatant from engineered MSC cultures wascollected and assayed by Western blot with anti-TAG antibody directed tothe bispecific protein. To test the capacity of engineered MSCs to killBCMA+ myeloma cells in the presence of naïve CD3+ T-cells, supernatantfrom MSC cultures were collected and added to co-cultures of BCMA+myeloma cell line expressing green fluorescent protein (MM.1S-GFP) andunlabeled bystander CD3+ T-cells. T-cells were collected from healthyvolunteers by Ficoll separation of mononuclear cells followed by CD3+bead positive selection. Aliquots of 50,000 MM.1S-GFP tumor cells wereplaced in wells of a 96-well plate, and between 2,500 to 50,000 T-cellswere added to each well to obtain various effector:target ratios (i.e.,ratios of T cells to BCMA+ myeloma cells) that were between about 1:1and 1:20. 100 μL of MSC supernatant was then added to experimentalwells. Negative controls included 100 μL of supernatant collected fromuntransfected MSCs, MM1.S tumor cells alone in the absence of T-cells,or T-cells alone in the absence of MM1.S tumor cells. Followingovernight incubation, propidium iodide was used to stain dead cells.Viable target cells were identified, and cell density was determined byflow cytometry. The degree of myeloma cell killing by engineered MSCsupernatant was calculated by comparison to the number of myeloma cellsin wells concurrent control wells that did not contain engineered MSCsupernatant.

It is expected that bifunctional anti-BCMA bispecific antibody, DNASE1and DNASE1L3 transfected MSCs will express and secrete their respectivefunctional proteins constitutively over the course of at least 24 hours.It is further expected that engineered MSCs will maintain the capacityto express and secrete functional anti-BCMA bispecific antibody, DNASE1and DNASE1L3 following freeze/thaw.

Thus, MSCs can be transfected with the inventive saRNA sequence toexpress and secrete functional anti-BCMA bispecific antibody, DNASE1 andDNASE1L3 protein. The result of this process is inventive, i.e.,bifunctional anti-BCMA bispecific antibody and DNase-secreting MSCs thatare useful for therapeutic administration, e.g., to a person affected byautoimmune diseases, e.g. generalized myasthenia gravis.

Example 8: Direct Comparison of Different Cells Types for Use in thePresent Invention

The following example describes a direct, head-to-head comparison of thechromatin-degrading activity of four different human cell types, eachmodified by identical methods to secrete a combination of DNASE1 andDNASE1L3. The study showed that MSCs were qualitatively superior to CD4+T cells, CD8+ T cells, and NK cells.

Umbilical MSCs were expanded in 2D vessels with standard culture medium.Naïve human CD4+ or CD8+ T cells were activated (anti-CD3) and expandedas known in the art. NK cells were obtained by negative-CD3 selectionand cultured in medium supplemented with IL-15, and then selectivelyexpanded by addition of mitomycin-treated K562 cells, as known in theart. The experimental operators had experience with culture of all ofthese cell types. Upon expansion, the above cells showed viability.

Comparable samples of the four aforementioned cell types (in each case10⁶ cells) were electroporated by identical methods with either of thefollowing:

-   -   a. sterile water (control); or    -   b. 2 μg of mRNA encoding Green Fluorescent Protein (GFP); 2 μg        of mRNA encoding DNASE1 (i.e., SEQ ID NO: 1); and 2 μg of mRNA        encoding DNASE1L3 (i.e., SEQ ID NO: 2), wherein substantially        all of the uridines were substituted with pseudouridine.        The Purpose of the GFP was to Verify that Electroporation and        Viability were Consistent Across the Four Aforementioned Cells        Types.

Transfected cells were plated with 2 mL culture medium per well.Supernatants were harvested 24 hours after transfection, and cells wereharvested for phenotyping.

Cells were phenotyped by flow cytometry. The MSC cell sample was >90%CD90+CD105+. The CD4+ T cell sample was >95% CD4+. The CD8+ cell samplewas >86% CD8+. The NK cell sample was 50% CD56+CD3−. Samples of all fourcell types were >90% GFP+.

The supernatant samples were tested with a chromatin digestion(degradation) assay as described in Example 1, supra. The results areshown in the gel electrophoresis of FIG. 1 . Where “no supernatant” wasincluded in the digestion assay, the chromatin was not degraded(negative controls). Likewise, in “untransfected” cells—i.e., each ofMSCs, CD4+ T cells, CD8+ T cells, and NK cells—chromatin was notdegraded (negative controls).

For cells transfected with mRNA to secrete a combination of DNASE1 andDNASE1L3, as described hereinabove, different cell types were associatedwith different levels of chromatin-degrading activity. CD8+ T cells hadthe least activity, followed by CD4+ T cells, then NK cells. Each of theforegoing cell types provided only partial chromatin degradation. MSCs,on the other hand, provided complete chromatin degradation, withsubstantially all degradation products at 180 bp or less.

Thus, in a direct head-to-head comparison of different cell typesmodified with mRNA to express a combination of DNASE1 and DNASE1L3, MSCsprovided superior chromatin-degrading activity. Furthermore, the MSCswere qualitatively superior to the other cell types tested, because onlythe MSCs provided for complete chromatin digestion.

Example 9: Synergistic Activity of Cells Secreting a Combination ofDNASE1 and DNASE1L3 Proteins

The following example shows that MSCs modified by mRNA electroporationto co-express both DNASE1 and DNASE1L3 possessed chromatin-degradingactivity that was synergistically and qualitatively superior to MSCsmodified to express either enzyme alone.

Umbilical MSCs were expanded in 2D vessels with standard culture medium,and 1×10⁶ cells were electroporated with water or mRNA as follows:

-   -   a. sterile water (control);    -   b. 2 μg of mRNA encoding DNASE1 (i.e., SEQ ID NO: 1);    -   c. 2 μg of mRNA encoding DNASE1L3 (i.e., SEQ ID NO: 2), wherein        substantially all of the uridines were substituted with        pseudouridine; or    -   d. 2 μg of mRNA encoding DNASE1 (i.e., SEQ ID NO: 1) and 2 μg of        mRNA encoding DNASE1L3 (i.e., SEQ ID NO: 2), wherein        substantially all of the uridines were substituted with        pseudouridine.

Transfected cells were plated into 6-well plates supplemented with 2 mLculture medium per well. The supernatant was harvested from each well 24hours after transfection, the wells were washed, and 2 mL culture mediumwas added to each well. The supernatant was harvested from each well 5days after transfection, and these supernatant samples were tested witha chromatin digestion (degradation) assay as described in Example 1,supra.

The results of the chromatin digestion assay are shown in FIG. 2 . Wherethe MSCs were unmodified (condition “a”, negative control), chromatinwas not degraded. Where the MSCs were modified to express DNASE1(condition “b”), chromatin was only partly degraded; the products ofdegradation were highly variable and indicated degradation atnonspecific sites, as the lane showed a smear indicating a panoply ofDNA/chromatin fragment sizes. Where the MSCs were modified to expressDNASE1 and DNASE1L3 (the latter by means of a pseudouridine-substitutedmRNA, condition “c”), chromatin was only partially degraded, but in adifferent pattern from that of condition “b”; here, a laddering patternwas seen wherein the fragments were multiples of 180 bp, e.g., 180, 360,540, 720, and 900 bp.

Where the MSCs were modified to express both DNASE1 and DNASE1L3(condition “d”), a surprising result occurred that was not onlysynergistic, but also qualitatively distinct from either condition “b”or condition “c.”. The DNA was almost completely digested to fragmentsof 180 bp.

Thus, MSCs modified by mRNA electroporation to co-express both DNASE1and DNASE1L3 possessed chromatin-degrading activity that wassynergistically and qualitatively superior to MSCs modified to expresseither of those enzymes alone.

Example 10: Characterization of GR-17, Human Mesenchymal Stem CellsElectroporated with mRNA Encoding Human DNASE1 and DNASE1L3

A series of six related in vitro studies was conducted to determine themRNA expression, protein expression, and activity of GR-17, humanmesenchymal stem cells (MSCs) electroporated with mRNA encoding humanDNASE1 and DNASE1L3.

Human MSCs derived from either adult bone marrow or umbilical cord werepurchased from RoosterBio, Inc. (Frederick, Md.). Serum-free MSC growthmedia (e.g., RoosterNourish) was purchased from RoosterBio. Cells werethawed and maintained in culture at 37° C. Cells were split byincubating in trypsin (Sigma) for 10 min, harvesting, and re-plating ata maintenance ratio for continued cell growth. Neutrophils weremaintained in ExCellerate media (R&D Systems) supplemented with 100×GlutaMax (Gibco).

Time Quantitative PCR was performed to evaluate level of mRNAtranscripts. mRNA was extracted from cells using an RNEasy kit (Qiagen)according to the manufacturer's instructions and quantitated byabsorption at A260 using a Nanodrop spectrophotometer (ThermoFisher).First-strand cDNA was generated using Superscript IV reversetranscriptase (ThermoFisher) and real-time quantitative PCR wasperformed using a SYBR green PCR 2×master mix (ThermoFisher) andgene-specific primers for either DNASE1 (forward-AGCTGGCTAGCTCTAAAGAAGC(SEQ ID NO: 25); reverse-TCTCCGAATGTCTGGATATTAAAGGC (SEQ ID NO: 26)) orDNASE1L3 (forward-AAGCAACAGCGTCTTCGAC (SEQ ID NO: 27);reverse-ATCTTTGTAGTCAGAGCCGCC (SEQ ID NO:28)). Amplification wasperformed on a MX3005P thermal cycler (Stratagene). Quantitation of mRNAwas determined by comparison with standard curves generated using knownquantities of plasmid DNA for DNASE1 or DNASE1L3.

To perform Western blots, 30 μL of GR-17 or control supernatant wasadded to 10 μL of 4× Laemmli loading buffer with (reducing) or without(non-reducing) 2-mercaptoethanol and denatured at 70° C. for 5 minutes.SDS-PAGE was performed using 4-20% Tris-glycine gradient gels andproteins were transferred onto PVDF membranes for Western blotting.Following blocking with 5% milk, blots were probed with mouse monoclonalanti-DNASE1 (B-4 clone, Santa Cruz sc-376207) followed by goatanti-mouse HRP (Abcam ab97040). For detection of DNASE1L3, 1 ml MSCcontrol cell or GR17-transfected cell culture medium at 24-hour postelectroporation, was used to pull down with 50 ul Heparin Sepharose. Theresin was then washed twice with 1 ml 10 mM Tris HCL pH 7.5 and mixedwith 4× Laemmli loading buffer with 2-mercaptoethanol and denatured at70° C. for 5 minutes and proteins transferred to PVDF membrane as above.DNASE1L3 was detected with rabbit polyclonal anti-DNASE1L3 antibody(Sigma SAB2107648) followed by goat anti-rabbit HRP antibody (Abcamab7090). The antibodies were detected by addition of Radiance Q (AzureBiosystems) and imaging of chemiluminescence using a C280 Imager (AzureBiosystems). The specificity and sensitivity of the Western blottingwere evaluated by spiking culture media with or without referencecontrol proteins for DNASE1 (Abcam ab73430) or DNASE1L3 (FLAG-tagged,produced using Origene RC205611; or GST-tagged Abcam ab238220), andcomparison of protein bands with a Precision Plus Dual Color referenceladder (Biorad 1610374).

To prepare chromatin, MM1S cells (human myeloma cell line) werecentrifuged, and the pellet was disrupted by gentle flicking andresuspended in 4° C. lysis buffer (0.5% Triton X-100 in 10 mM Tris pH7.8 with 150 mM NaCl) at 100M/mL. The cells were gently vortexed andincubated on ice for 10 min. The nuclei were pelleted by centrifugationand resuspended in ice-cold PBS at 10M/mL. An equal volume of glycerolwas added, and the nuclei were stored in aliquots at −80° C.

To prepare NETs, neutrophil polymorphonuclear granulocytes (PMNs) wereisolated from whole blood within 2 hours after collection withHistopaque-1119 (Sigma) and Lymphoprep (Stemcell Technologies) accordingto the manufacturers' instructions. 10 mL of red cell lysis buffer (ACKbuffer, Sigma) was added and the solution incubated at room temperaturefor 6 to 8 minutes. The cells were washed twice and resuspended inserum-free media at the desired concentration.

To induce NETosis, freshly isolated neutrophils were resuspended inserum-free neutrophil media (see above for culture conditions) and 100nM PMA (Sigma-Aldrich). The desired number of cells (e.g. 10⁵) was addedto each well of a 96-well flat-bottom CellBind plate (Corning) in 100 uLand the cells were incubated at 37° C. Following overnight incubation,the plates were centrifuged at 200×g for 3 minutes. NET formation wasconfirmed by adding a 1:500 dilution of Sytox Green (Life Technologies)per manufacturer's protocol and examined under fluorescent microscopyusing a Cytation-5 Imaging system (BioTek). The plates were stored at 4°C. for up to 1 month to be used in NET digestion assays.

DNA degradation assays were performed using chromatin (15K lysednuclei), NETs (equivalent of 100K neutrophils) or naked DNA (1 μgpurified R′ plasmid) using the following procedure. A 2×master mix ofDNA and buffer (including 10×DNASE1 digestion buffer, New EnglandBiolabs NEB #B0303S) was prepared on ice and added to 10 μL of undilutedsample or sample diluted in nuclease-free H₂O. Recombinant human DNASE1(Abcam ab73430) and recombinant human DNASE1L3 (produced in 293 cellsusing Origene RC205611) were added to separate reactions as controls.DNA digestion was performed by incubating samples for 30 minutes at 37°C. in a water bath. The reaction was stopped by adding 1 μL of 100 mMEDTA to each tube, and the protein was digested by the addition of 2 μlof 20 mg/mL of Proteinase K. The reaction was incubated for another 20minutes at 37° C. The nucleic acid was extracted by addition of an equalvolume of Phenol:Chloroform:Isoamyl Alcohol 25:24:1 (Sigma), gentlevortexing and centrifugation at 16,000×g for 10 minutes at roomtemperature. The aqueous layer was added to 6×DNA loading dye andresolved on a 1.5% agarose gel containing Gel-Red nucleic acid stain(Biotium) in comparison with a 100 bp DNA ladder (NEB N04675). Imageswere captured using a C280 Imager (Azure Biosystems).

Study GR17-1 visualized the capacity of GR-17 cells to degrade largequantities of NETs that were visible macroscopically. 1.5×10⁶ MSCs weretransfected with DNASE1 and DNASE1L3 mRNA or irrelevant control mRNA(R′; see IND 19050 construct) synthesized in identical fashion.Transfected cells were plated on a 6-well CellBind plate in 1 ml ofRoosterBio media for 48 hours. Supernatants were collected and addeddirectly to slides containing large quantities of visible, viscous NETsgenerated from 4×10⁶ neutrophils, and the slides were incubated on a 37°C. heat block. The slides were videographed and photographic clips werecaptured at various timepoints. GR-17 supernatant, but not control MSCsupernatant, degraded large quantities of NETs (FIG. 3 ). Partialdegradation was noticeable within the first 5 minutes. Degradation wascomplete within about 10 minutes. It was concluded that GR-17 degradeslarge, visible quantities of NETs within minutes.

Study GR17-2 determined the presence of mRNA, DNase secretion, andNET-degrading capacity of GR-17 cells over 1 week in cell culture. 10million human MSCs were electroporated using 100 μL cuvettes with 1 μgof mRNA encoding human DNASE1 and DNASE1L3, cultured for 2 hours,frozen, stored overnight at −80° C., thawed, and cultured at 37° C. forup to 6 days in RoosterNourish media (RoosterBio, Frederick, Md.). Celllysates were collected to detect mRNA by RT-qPCR analysis. Supernatantswere collected to detect DNase protein by Western blot. Supernatant wasalso used to assay enzyme activity by degradation of naked DNA,chromatin, and neutrophil extracellular traps (NETs). GR-17 cellsexpressed DNase mRNA that waned off over days (FIGS. 4A-4B). DNASE1 mRNAwas detectable at higher copy numbers and for a longer time comparedwith DNASE1L3 mRNA (FIG. 4A vs 4B; compare peak copies per ng total mRNAof 30,000 vs. 950 and compare undetectable mRNA at 6 days vs 2 days).Waning mRNA translated to reduced protein expression over time (FIGS.5A-5B); and peak expression of both proteins was at Day 1.DNASE1-protein expression was reduced over days and was only faintlydetectable at Day 6 (FIG. 5A). DNASE1L3 protein was detectable at Day 1only (FIG. 5B). Waning protein expression translated to reducedenzymatic activity over time (FIGS. 6A-6D). peak activity was measuredat Day 1 and waned over 6 days as measured by the capacity ofsupernatant to degrade cell-free naked DNA, chromatin, and NETs (FIGS.6A-6D). A representative fluorescent micrograph of NETs is provided inFIG. 6D. It was concluded that GR-17 expresses DNASE1 and DNASE1L3protein that degrades NETs. Activity wanes over about 1 week likely dueto transient expression of mRNA.

Study GR17-3 assessed the dose-dependent NET-degrading capacity of GR-17cells when cultured directly with exogenous NETs, which may betterreflect conditions in the human lung with ARDS. Human MSCs weretransfected with DNASE1 mRNA and DNASE1L3 mRNA (GR-17) or water (controlMSCs) and cultured overnight in 96-well plates. Media was removed andfresh media containing 20 μg/mL of naked DNA or 100 μL NETs (generatedfrom 100,000 neutrophils incubated with 100 nM PMA) was added directlyonto MSCs. The cells were cultured overnight and assayed for degradationof DNA or NETs as described above. Control MSCs did not degrade eithercell-free naked DNA or NETs when incubated in culture overnight. TheGR-17 cells degraded naked DNA (FIG. 7A) and NETs (FIGS. 7B, 7C) in adose-dependent manner. GR-17 viability was unaffected in the presence ofexogenous NETs. A representative fluorescent micrograph of NETs isprovided in FIG. 7C. It was concluded that GR-17 degrades cell-freenaked DNA and NETs in a dose-dependent manner.

Study GR17-4 assessed the time-dependent NET-degrading capacity of GR-17cells when cultured directly with exogenous NETs, which may betterreflect conditions in the human lung with ARDS. The GR-17 cells orcontrol MSCs were plated in a 96-well plate at 100,000 per well and in a2-fold dilution series to 200 per well. 24 hours after plating,supernatants were removed and adherent GR-17 or control MSC monolayerswere washed twice with tissue culture media. Either NETs induced from10⁵ neutrophils in 100 ul of ExCellerate media or naked plasmid DNA (5μg/100 μL culture) were added directly onto cultures and co-cultured forup to 48 hours. Supernatants were analyzed for presence of NETs usingfluorescence microscopy with Sytox green (ThermoFisher) anddouble-stranded DNA using a Quant-iT™ dsDNA assay (high sensitivity;ThermoFisher) and fluorometry on a Cytation-5 plate reader (BioTek).GR-17 digested NETs over 2 days without loss in activity (FIG. 8 ). NETdigestion was detectable as early as 2 h and increased to completedigestion at about 14 hours. It was concluded that GR-17 digests NETs ina time-dependent manner.

To confirm the specific activity of each enzyme, Study GR17-5 determinedthe capacity of DNASE1 or DNASE1L3 to digest chromatin over time. 10million human MSCs were transfected with 1 μg of mRNA encoding eitherhuman DNASE1 or human DNASE1L3, cultured for 2 hours, frozen, storedovernight at −80° C., thawed, and cultured for up to 1 week.Supernatants were collected at the indicated timepoints (see FIG. 9 )and their chromatin-digesting activity was assayed as described above.DNASE1 and DNASE1L3 were each capable of degrading chromatin andmaintained activity for at least 3 days (FIG. 9 ). DNASE1chromatin-digesting activity was detectable up to Day 7 while DNASE1L3activity waned after about 3 days. Differential activity between DNASE1and DNASE1L3 was consistent with differences mRNA and protein expressionbetween these enzymes (see Study GR17-1). It was concluded that HumanMSCs transfected with DNASE1 mRNA or DNASE1L3 mRNA can degrade chromatinfor at least 3 days.

Study GR17-6 determined the capacity of GR-17 to degrade DNA and NETs inthe presence of human serum. GR-17 was frozen, thawed, and incubated inthe presence of NETs overnight (0.1×10⁶ cells/well) in media with 0%,50% or 100% fresh off-the-clot serum collected from healthy volunteers.Serum incubation was done in replicates. GR-17 maintained itsNET-degrading capacity in the presence of human serum (FIG. 10 ). Theserum-alone exhibited NET-degradation. NET digestion was evident butincomplete in the presence of serum. GR-17 completely eliminated NETseven in the presence of 100% serum, indicating that serum does notinhibit the capacity of GR-17 to inhibit NETs. Results arerepresentative of serum collected from three different healthy donors.It was concluded that Human serum does not interfere with GR-17'scapacity to degrade NETs.

Therefore, GR-17 cells express mRNA and protein for DNASE1 and DNASE1L3.GR-17 shows potent activity to degrade cell-free naked DNA, chromatin(nuclei), and NETs in a time- and dose-dependent manner. GR-17 candegrade large (visible) amounts of NETs within 10 minutes.

Example 11: Production of Functional DNAse-Secreting NK Cells from anInventive Combination of mRNA Constructs

The following example is of human DNAse-secreting NK cells produced byintroduction of an inventive combination of mRNA constructs that encodehuman DNASE1 and DNASE1L3.

Two separate mRNA constructs, corresponding respectively to SEQ ID NO: 1(DNASE1) and SEQ ID NO: 2 (DNASE1L3), were generated by in vitrotranscription. The in vitro transcription was performed by amplificationof the double-stranded DNA template from DNA plasmids by PCR followed byin vitro transcription using T7 RNA polymerase. An additionalpolyadenine tail of about 100 adenine nucleotides was addedenzymatically to each mRNA. A 7-methylguanosine cap was incorporated atthe 5′ end of each mRNA during the co-transcriptional RNA synthesis.

The first mRNA construct comprised, from 5′ to 3′: a 5′ cap; a 5′ UTRdescribed as SEQ ID NO: 23; a Kozak sequence described in SEQ ID NO: 20;a sequence of SEQ ID NO:1; a 3′ UTR described as SEQ ID NO: 24, and a 3′polyadenine tail of 180 adenine units or more.

The second mRNA construct comprised, from 5′ to 3′: a 5′ cap; a 5′ UTRdescribed as SEQ ID NO: 23; a Kozak sequence described in SEQ ID NO: 20;a sequence of SEQ ID NO:2; a 3′ UTR described as SEQ ID NO: 24, and a 3′polyadenine tail of 180 adenine units or more.

To prepare NK cells, NK cells were isolated from whole blood byisolation of peripheral blood monocytes (PBMC) by centrifuge gradientfollowed by CD56+ magnetic bead positive selection to obtain >98% pureNK cells that are >90% viable. These NK cells were expanded byincubation at 37° C. with 5% CO₂ in the presence of K562 feeder cellsand supplemented with IL15 for 7 to 10 days. The cells are resuspendedin transfection buffer and simultaneously transfected with a mixture ofthe two aforementioned mRNA constructs by electroporation (4DNUCLEOFECTOR®, Lonza) according to manufacturer's instructions. Cellsare then returned to culture in a standard medium containing IL-15 forovernight incubation prior to analysis for viability and activityaccording to the methods described below.

NK cells obtained from the above-described process were tested forviability and NET-degrading capacity. Viability was determined by anautomated cell counter (Auto2000, Nexcelom Biosciences) followingstaining with ViaStain™ (acridine orange and propidium iodide) accordingto the manufacturer's protocol. To test NET-degrading capacity oftransfected NK cells, culture supernatants were first incubated withNETs generated from phorbol myristate acetate-activated humanneutrophils. Neutrophils were isolated from whole anti-coagulated bloodby density grade centrifugation. Recombinant human DNASE1 and DNASE1L3were used as positive controls. The amount of NET DNA released wasdetermined by addition of picogreen (Invitrogen), a DNA fluorescencedye, to the mixed culture and then quantified by fluorescencespectrometry.

NK cells transfected with the above-described constructs expressed andsecreted functional DNASE1 and DNASE1L3 constitutively over the courseof at least 24 hours.

Thus, NK cells can be transfected with the inventive combination of mRNAsequences to express and secrete functional DNASE1 and DNASE1L3 protein.The result of this process is inventive, DNase-secreting, NK cells thatare useful for therapeutic administration, e.g., to a person affected byARDS.

Example 12: Production of Functional DNAse-Secreting MSCs from anInventive Combination of mRNA Constructs

The following example is of human DNAse-secreting MSCs produced byintroduction of an inventive combination of mRNA construct that encodehuman DNASE1 and DNASE1L3.

Two separate mRNA constructs, corresponding respectively to SEQ ID NO: 1(DNASE1) and SEQ ID NO: 2 (DNASE1L3) were generated by in vitrotranscription. The in vitro transcription was performed by amplificationof the double-stranded DNA template from DNA plasmids by PCR with a 5′primer and a 3′ primer that contains 180 additional thymidinenucleotides prior to the template-binding sequence. The PCR product wasused for in vitro transcription using T7 RNA polymerase. An additionalpolyadenine tail of about 60 adenine nucleotides was added enzymaticallyto each mRNA. A 7-methylguanosine cap was incorporated at the 5′ end ofeach mRNA during the co-transcriptional RNA synthesis.

The first mRNA construct comprised, from 5′ to 3′: a 5′ cap; a 5′ UTRdescribed as SEQ ID NO: 23; a Kozak sequence described in SEQ ID NO: 20;a sequence of SEQ ID NO:1; a 3′ UTR described as SEQ ID NO: 24, and a 3′polyadenine tail of 180 adenine units or more.

The second mRNA construct comprised, from 5′ to 3′: a 5′ cap; a 5′ UTRdescribed as SEQ ID NO: 23; a Kozak sequence described in SEQ ID NO: 20;a sequence of SEQ ID NO:2; a 3′ UTR described as SEQ ID NO: 24, and a 3′polyadenine tail of 180 adenine units or more.

To prepare MSCs from RNA constructs, MSCs isolated from human umbilicalcord were purchased from ATCC, thawed and expanded in T75 flasks byculture at 37° C. with 5% CO₂ in α-DMEM medium supplemented with 10%human serum. The cells were then passaged upon reaching 80% confluency.The cells were resuspended in P3 transfection buffer (Lonza) andsimultaneously transfected with a mixture of the two aforementioned mRNAconstructs by electroporation (4D NUCLEOFECTOR®, Lonza) according tomanufacturer's instructions. Cells were then returned to culture incomplete medium for overnight incubation, then frozen at −80° C.Transfected cells were thawed and incubated for another 7 days in thepresence of complete medium and supernatant samples were collected andfrozen at 4 h, 1 day, 2 days, 3 days, 5 days and 7 days after thaw.Thawed cells and supernatants were assayed for viability and activityaccording to the methods described below.

MSCs obtained from the above-described process were tested forviability, DNASE1 and DNASE1L3 expression, and DNA- andchromatin-degrading capacity. Viability was determined by flow cytometryon a GUAVA® EASYCYTE® 12HT Flow cytometer (EMD Millipore). To testviability, a sample of the MSCs was mixed with propidium iodide and runon the flow cytometer with electronic gating on fluorescence in the nearinfrared channel. To test expression of DNASE1 and DNASE1L3,commercially available DNASE1 and DNASE1L3 kits (i.e., Abbexa) were usedto assay supernatants of cultured MSCs according the manufacturers'instructions. To test DNA degrading capacity of transfected MSCs,culture supernatants were tested with a fluorometric DNASE1 assay kit(i.e., AbCam) according to the manufacturer's instructions. To testchromatin degrading capacity of transfected MSCs, culture supernatantswere first incubated with NETs generated from phorbol myristateacetate-activated human neutrophils. Neutrophils were isolated fromfresh apheresis product by density grade centrifugation. Recombinanthuman DNASE1 and DNASE1L3 were used as positive controls. The amount ofNET DNA released was determined by adding picogreen (Invitrogen), a DNAfluorescence dye, to the mixed culture and then quantified byfluorescence spectrometry.

The MSCs transfected with the above-described constructs express andsecrete functional DNASE1 and DNASE1L3 constitutively over the course ofat least 24 hours. MSCs maintain the capacity to express and secretefunctional DNASE1 and DNASE1L3 following freeze/thaw.

Thus, MSCs can be transfected with the inventive combination of mRNAsequences to express and secrete functional DNASE1 and DNASE1L3 protein.The result of this process is inventive, i.e., DNase-secreting MSCs thatare useful for therapeutic administration, e.g., to a person affected byARDS.

Example 13: In Vivo Safety and Efficacy of Functional DNAse-SecretingMSCs in a Mouse Model of ALI

DNAse-secreting MSCs produced from inventive RNA constructs comprisingthe nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, were testedin an Acute Lung Injury (ALI) animal model of ARDS. In this model, ALIwas first induced by intratracheal administration of lipopolysaccharide(LPS), and MSCs were then administered by intravenous injection.

DNAse-secreting MSCs were prepared by transfection of the inventive RNAconstructs, as discussed in Example 12. 8-12 week old C57BL/6 mice wereanesthetized and administered 2 mg/kg of LPS solution by anintratracheal administration. At 12 hours after LPS administration, micewere randomized to receive intravenous vehicle only (negative control),1.5×10⁶ untransfected MSCs (negative control), or 0.25×10⁶ or 1×10⁶DNAse-secreting MSCs. 4 to 7 animals were assigned to each group.

24 hours after MSC administration, 3 mice from each group wereanesthetized and bronchoalveolar lavage fluid (BALF) was collected with1 mL phosphate buffered saline (PBS) solution. Mice were thensacrificed; blood and lung tissues collected.

Total cell number and infiltration of specific cell types (i.e.,macrophages, neutrophils and lymphocytes) in BALF were analyzed withflow cytometry on a Guava EasyCyte HT flow cytometer (Luminex).Nucleated cells were identified by Forward and Side scatter propertiesand concentrations were determined by comparison with known numbers ofcounting beads using 123count eBeads (Invitrogen). The remaining samplewas used for cell-free DNA measurement and detection of NET-specificmarkers in sandwich ELISA assays, specifically Myeloperoxisdase(MPO)-DNA complexes, neutrophil elastase (NE), and citrullinated histone(CitH3).

Serum was collected from whole blood and frozen down for subsequentanalysis of NET-specific markers in sandwich ELISA assays similar toBALF.

Lungs were harvested, the left lungs were flashed frozen, and the rightlungs were fixed and fixed in 4% formalin solution for subsequentpathologic analysis.

5 days after LPS administration, the remaining mice were sacrificed,blood and lung tissue was collected and processed as above.

All animals tolerated treatment and recovered following a transient ALIbetween Days 1 and 3 with the exception of one mouse that died prior torandomization and treatment on Day 1. There were no differences betweenthe two Descartes-30 groups and control animals with respect to clinicalobservations, daily weights, organ weights, and overall survival.

Example 14: Production of Functional DNAse-Secreting MSCs from anInventive Combination of mRNA Constructs with or without Pseudouridine

The following example is of human DNAse-secreting MSCs produced byintroduction of an inventive combination of mRNA construct that encodehuman DNASE1 and DNASE1L3, where such mRNAs are enriched or not enrichedin pseudouridine.

MSCs were prepared and tested according to the methods Example 12 above,from mRNAs that were wild-type (U) with respect to uridine, or for whichthe uridine positions were substantially substituted with pseudouridine(ψ).

FIG. 11 shows the results of a chromatin degradation assay with MSCstransfected with wild-type (U) or pseudouridine (ψ) DNase1 or DNase1L3mRNA and cultured as described. Between timepoints, e.g., day 0 to day 1(D1), supernatants were collected and used in chromatin digestionassays.

Supernatants from MSC transfected with DNase1L3 mRNA consistentlyresulted in greater chromatin digestion, signifying higher production ofDNase1L3, when the mRNA was encoded using pseudouridine rather thanuridine. However, modification of the DNase1 rRNA with pseudouridine didnot increase its level of activity.

Thus, substitution of pseudouridine for uridine in mRNA encodingDNase1L3 significantly improved chromatin digestion, whereassubstitution of pseudouridine for uridine in mRNA encoding DNase1 didnot improve chromatin digestion.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation, many equivalents of the embodimentsdescribed herein. The scope of the present disclosure is not intended tobe limited to the above description, but rather is as set forth in theappended claims.

Articles such as “a,” “an,” and “the” may mean one or more than oneunless indicated to the contrary or otherwise evident from the context.Claims, embodiments, or descriptions that include “or” between two ormore members of a group are considered satisfied if one, more than one,or all of the group members are present, unless indicated to thecontrary or otherwise evident from the context. The disclosure of agroup that includes “or” between two or more group members providesembodiments in which exactly one member of the group is present,embodiments in which two or more members of the group are present, andembodiments in which all of the group members are present. For purposesof brevity those embodiments have not been individually spelled outherein, but it will be understood that each of these embodiments isprovided herein and may be specifically claimed or disclaimed.

It is to be understood that the invention encompasses all variations,combinations, and permutations in which one or more limitation, element,clause, or descriptive term, from one or more of the claims or from oneor more relevant portion of the description, is introduced into anotherclaim. For example, a claim that is dependent on another claim can bemodified to include one or more of the limitations found in any otherclaim that is dependent on the same base claim. Furthermore, where theclaims recite a composition, it is to be understood that methods ofmaking or using the composition according to any of the methods ofmaking or using disclosed herein or according to methods known in theart, if any, are included, unless otherwise indicated or unless it wouldbe evident to one of ordinary skill in the art that a contradiction orinconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that every possible subgroup of the elements is alsodisclosed, and that any element or subgroup of elements can be removedfrom the group. It is also noted that the term “comprising” is intendedto be open and permits the inclusion of additional elements or steps. Itshould be understood that, in general, where an Claim, product, ormethod is referred to as comprising particular elements, features, orsteps, embodiments, products, or methods that consist, or consistessentially of, such elements, features, or steps, are provided as well.For purposes of brevity those embodiments have not been individuallyspelled out herein, but it will be understood that each of theseembodiments is provided herein and may be specifically claimed ordisclaimed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and/or the understanding of one of ordinary skill in the art,values that are expressed as ranges can assume any specific value withinthe stated ranges in some embodiments, to the tenth of the unit of thelower limit of the range, unless the context clearly dictates otherwise.For purposes of brevity, the values in each range have not beenindividually spelled out herein, but it will be understood that each ofthese values is provided herein and may be specifically claimed ordisclaimed. It is also to be understood that unless otherwise indicatedor otherwise evident from the context and/or the understanding of one ofordinary skill in the art, values expressed as ranges can assume anysubrange within the given range, wherein the endpoints of the subrangeare expressed to the same degree of accuracy as the tenth of the unit ofthe lower limit of the range.

In addition, it is to be understood that any particular Claim of thepresent invention may be explicitly excluded from any one or more of theclaims. Where ranges are given, any value within the range mayexplicitly be excluded from any one or more of the claims. Any Claim,element, feature, application, or aspect of the compositions and/ormethods of the invention, can be excluded from any one or more claims.For purposes of brevity, all of the embodiments in which one or moreelements, features, purposes, or aspects is excluded are not set forthexplicitly herein.

1-20. (canceled)
 21. A mammalian cell modified to express: at least oneDNAse enzyme; and at least one anti-BCMA protein; wherein the cellcauses reduction of: neutrophil extracellular traps; and BCMA+ cells.22. The cell of claim 21, wherein the cell is a mesenchymal stem cell.23. The cell of claim 21, wherein the cell is a T cell.
 24. The cell ofclaim 23, wherein the cell is a CD8+ cell.
 25. The cell of claim 21,wherein the anti-BCMA protein comprises a monoclonal antibody that bindsBCMA.
 26. The cell of claim 22, wherein the anti-BCMA protein comprisesa monoclonal antibody that binds BCMA.
 27. The cell of claim 23, whereinthe anti-BCMA protein comprises a monoclonal antibody that binds BCMA.28. The cell of claim 24, wherein the anti-BCMA protein comprises amonoclonal antibody that binds BCMA.
 29. The cell of claim 21, whereinthe cell is modified by introduction of mRNA.
 30. The cell of claim 22,wherein the cell is modified by introduction of mRNA.
 31. The cell ofclaim 23, wherein the cell is modified by introduction of mRNA.
 32. Thecell of claim 24, wherein the cell is modified by introduction of mRNA.33. The cell of claim 25, wherein the cell is modified by introductionof mRNA.
 34. The cell of claim 26, wherein the cell is modified byintroduction of mRNA.
 35. The cell of claim 27, wherein the cell ismodified by introduction of mRNA.
 36. The cell of claim 28, wherein thecell is modified by introduction of mRNA.
 36. The cell of claim 29,wherein the introduction of mRNA is by electroporation.
 37. The cell ofclaim 30, wherein the introduction of mRNA is by electroporation. 38.The cell of claim 31, wherein the introduction of mRNA is byelectroporation.
 39. The cell of claim 32, wherein the introduction ofmRNA is by electroporation.
 40. The cell of claim 33, wherein theintroduction of mRNA is by electroporation.
 41. The cell of claim 34,wherein the introduction of mRNA is by electroporation.
 42. The cell ofclaim 35, wherein the introduction of mRNA is by electroporation. 43.The cell of claim 36, wherein the introduction of mRNA is byelectroporation.